Compound having affinity with calcified tissue

ABSTRACT

The present invention provides a compound excellent in accumulation on calcified tissues and ensuring rapid excretion into urine as well as rapid clearance from blood, useful as a diagnostic agent and a therapeutic agent, etc. The compound is a compound having affinity with a calcified tissue represented by the following formula: (AC) a -MC-(LI) b . In the formula, MC is a mother nucleus and represents a residue of a compound having a plurality of functional groups selected from the group consisting of an amino group, an amide group, a hydroxyl group, a thiol group, a thioether group, a sulfonyl group, a phosphonyl group, an aldehyde group, a carboxyl group, a carbonyl group, a halogen, and a cyano group; AC is a group having affinity with a calcified tissue; LI is a ligand for binding to a metal atom; and a is an integer of 1 or more and b is 0 or an integer of 1 or more.

TECHNICAL FIELD

The present invention relates to a compound having high affinity with acalcified tissue and exhibiting rapid excretion into urine, and usethereof as a diagnostic agent, a therapeutic agent and the like.

BACKGROUND ART

In recent years, skeletal scintigraphy by nuclear medical technique isbecoming an important tool in diagnosing bone diseases of an initialstage. Imaging agents for bone scintigraphy are required to have highexcretability into urine and rapid clearance from blood and tissues aswell as high bone affinity, etc. so that the time from administeringagents till performing imaging can be shortened and scintigrams of highquality can be obtained. Today, phosphate compounds labeled with aradioisotope are used. At first, an inorganic polyphosphate labeled with99m-technetium was tried. However, the inorganic polyphosphate labeledwith 99m-technetium was problematic in that it was hydrolyzed tomonophosphate in an aqueous solution and accordingly the clearance fromblood was low.

In order to solve this problem, Yano et al. reported stannousTc-99m-ethane-1-hydroxy-1-diphosphonate (Tc-99m-HEDP) which is anorganic diphosphonate labeled with 99m-technetium (J. Nucl. Med. 14, 73,(1973) and U.S. Pat. No. 3,735,001 specification). Since this compoundexhibits relatively rapid clearance from blood, bone scintigraphyexamination can be conducted in a shorter time after administering theagent, and 99m-technetium-labeled phosphate compounds analogous toTc-99m-HEDP, i.e., 99m-technetium labeled organic diphosphonatecompounds such as methane diphosphonate (MDP) and hydroxymethanediphosphonate (HMDP) have been widely used to date. Althoughpreparations for bone scintigraphy using these compounds accumulate atthe site where osseous calcification proceeds and further shorten thetime from administering the agent till performing imaging, they stilltake about three hours after administration, which cannot be said to besufficiently short.

Generally, when a preparation for bone scintigraphy is slow in theclearance from blood and/or soft tissues and the excretion into urine,the time from administering the agent till performing imaging becomeslonger in order to improve contrast of image. It is considered thatpolymer structure of technetium-bisphosphonate complexes is one of thefactors that affect the clearance of technetium-labeled phosphatecompounds. Although an attempt to promote the clearance at an earlystage after administration by changing a bisphosphonate compound from apolymer structure to a monomolecular structure was made on abisphosphonate compound labeled with 123-iodine (WO89/11877), but theefficacy is not necessarily sufficient.

From the same viewpoint, various organic phosphonic acid compounds suchas bisphosphonic acid derivatives have also been proposed (JapanesePatent Laid-Open No. 59-205331, Japanese Patent Laid-Open No. 57-50928,Japanese Patent Laid-Open No. 63-500849, Japanese Patent Laid-Open2001-114792), but they are still required to be improved inexcretability in urine and blood clearance so that the time fromadministering the agent till taking an image can be shortened.

DISCLOSURE OF THE INVENTION

Therefore, one object of the present invention is to provide novelorganic phosphonic acid derivatives which can be compounds withexcellent affinity to calcified tissue but excreted into urine at highrate when they fail to accumulate in calcified tissue by designing thederivatives so that their organic phosphonic acid groups do not readilyform complexes and the molecular size of the compounds with excellentaffinity to calcified tissue is controlled, the other is to provide itsuse as a diagnostic agent, a therapeutic agent and the like.

The present inventors have conducted various studies on organicphosphonic acids and other compounds having affinity with a calcifiedtissue to attain the above-mentioned object, and have found thatcompounds which are represented by the following general formula:(AC)_(a)-MC-(LI)_(b) (wherein, MC is a mother nucleus, AC is a grouphaving affinity with a calcified tissue, and LI is a ligand for bindingto a metal atom; a is an integer of 1 or more; b is 0 or an integer of 1or more) and have a mother nucleus MC of a controlled molecular sizeexhibit excellent affinity with a calcified tissue while the compoundsthat fail to accumulate in calcified tissues exhibit high excretabilityinto urine, and thereby completed the present invention.

That is, according to one aspect of the present invention, a compoundhaving affinity with a calcified tissue represented by the followinggeneral formula:(AC)_(a)-MC-(LI)_(b)(wherein MC is a mother nucleus and represents a residue of a compoundhaving a plurality of functional groups selected from the groupconsisting of an amino group, an amide group, a hydroxyl group, a thiolgroup, a thioether group, a sulfonyl group, a phosphonyl group, analdehyde group, a carboxyl group, a carbonyl group, a halogen, and acyano group; AC is a group having affinity with a calcified tissue; LIis a ligand for binding to a metal atom; and a and b are integers of 1or more) is provided.

Here, the ligand LI can bind to a metal atom, and thus may form acomplex with a metal atom but does not need to form a complex. Since theLI moiety plays a central role in complex formation ability, and thusthe AC moiety does not readily form a complex compound, the presentcompounds exhibit a rapid clearance from the blood and/or soft tissueand also a rapid excretion into urine advantageously.

Preferable compounds of the present invention are represented by theformula: (AC)_(a)-MC-(LI)_(b) (wherein MC represents a residue of acompound selected from the group consisting of a monosaccharide, anoligosaccharide, an amino oligosaccharide, a cyclodextrin and asaccharide dendrimer; a and b are integers of 1 or more). AC in theabove formula is preferably a calcified tissue-affinity group whichcomprises a polyaspartic acid group, a polyglutamic acid group and anorganic phosphonic acid group, and more preferably it is an organicphosphonic acid group.

More preferable compounds of the present invention are represented bythe formula: (AC)_(a)-MC-(LI)_(b) (wherein MC represents a residue of acompound selected from the group consisting of an oligosaccharide, anamino oligosaccharide, a cyclodextrin and a saccharide dendrimer, andthe group AC having affinity with a calcified tissue is bonded to anyone of the constituent monosaccharides of the mother nucleus MC, and theligand LI for binding to a metal atom is bonded to a constituentmonosaccharide other than the above-mentioned constituentmonosaccharide; a and b are integers of 1 or more). A plurality of thecalcified tissue-affinity group AC or the ligand LI for binding to ametal atom may be bonded to the mother nucleus MC.

Furthermore, according to another aspect of the present invention, acompound having affinity with a calcified tissue represented by theformula: (AC)_(a)-MC (wherein MC is a mother nucleus and represents aresidue of a compound having a plurality of functional groups selectedfrom the group consisting of an amino group, an amide group, a hydroxylgroup, a thiol group, a thioether group, a sulfonyl group, a phosphonylgroup, an aldehyde group, a carboxyl group, a carbonyl group, a halogen,and a cyano group;

AC is a group having affinity with a calcified tissue; and

a is an integer of 1 or more) is provided. This compound is advantageousin that it has a mother nucleus MC of a controlled molecular size andexhibits excellent affinity with a calcified tissue by virtue of the ACwhile the compound which fails to accumulate in calcified tissuesexhibits high excretability in urine by virtue of the MC.

In the compound represented by the formula (AC)_(a)-MC, it is preferablethat the MC is a residue of a compound selected from the groupconsisting of a monosaccharide, an oligosaccharide, an aminooligosaccharide, a cyclodextrin and a saccharide dendrimer and the AC ispreferably a calcified tissues-affinity group comprising a polyasparticacid group, a polyglutamic acid group and an organic phosphonic acidgroup, and the AC is more preferably an organic phosphonic acid group.

According to a preferable embodiment, the compound of the presentinvention comprises a metal atom or an isotope of a halogen atom,carbon, oxygen, nitrogen, sulfur or phosphorus in at least one of themother nucleus MC, the calcified tissue-affinity group AC and the ligandLI. This embodiment is particularly preferable for use as a diagnosticagent.

Japanese Patent Laid-open No. 10-501218 discloses a 99m-technetiummono-, di- or polyphosphonate complex composition which has acomposition variable with conditions of heating by autoclaves,microwaves and the like. This is an attempt to improve aggravation ofthe clearance caused by the formation of a polymer structure of theradioactive metal labeled phosphate compound. In this method, however,co-existence of polyphosphonates having polymer structures is still notavoided.

On the other hand, the compound represented by the formula(AC)_(a)-MC-(LI)_(b) of the present invention is characterized in thatthe labeling function by a radioactive nuclide is assigned to the ligandLI, and the opportunity for bisphosphonic acid to participate in complexformation is reduced, thereby contemplating a more advantageousimprovement of accumulation to the bone. The same technical idea isapparently applicable to the calcified tissue-affinity group AC otherthan the bisphosphonic acid. The difference in the complex formationability between the ligand LI and the calcified tissue-affinity group ACcan be proved by using an analog of the compound of the presentinvention. The idea that a calcified tissue-affinity group AC forms nocomplex can be proved by selecting labeling conditions such asradioactive metal nuclides, concentration, pH and reducing agents. Forexample, it can be proved by a coexistence labeling method using DTPA orMAG3 together with HMDP.

In addition, the compound represented by the formula (AC)_(a)-MCaccording to the present invention has affinity with a calcified tissuein itself, and thus is useful not only as a therapeutic agent but alsoas a diagnostic agent since it can be labeled by allowing an isotope ofa metal atom or a halogen atom, carbon, oxygen, nitrogen, sulfur orphosphorus to be contained in at least one of the mother nucleus MC andthe calcified tissue-affinity group AC.

The use of diagnostic agents in diagnostic medicine has been rapidlyincreasing. Conventionally a composition or a substance labeled with aradioisotope has been administered to a living body in nuclear medicinediagnosis, wherein the radiation that the composition or substance emitsis detected by a scintillation camera and the distribution and behaviourof the composition or substance in the living body is expressednon-invasively as an image, and this technique has been used for earlydetection of various diseases and elucidation of pathologic conditions.The composition and substance labeled with a radioisotope are calledradioactive imaging agents, and those suitable for specific purposeshave been developed. Furthermore, in MRI diagnosis, contrast of tissuescan be enhanced by administering a composition or substance containing aparamagnetic metal species which increases the relaxation of surroundingprotons.

On the other hand, referring to therapeutic agents, it has been knownthat bisphosphonic acids have, by virtue of their affinity withcalcified tissues, an effect of inhibiting bone absorption and increaseof serum calcium level resulting from enhancement of the boneabsorption. Thus, they have been introduced to clinical practice asactive substances in drugs for treating diseases which morbid conditionis considered to have a significant relation with bone absorption, forexample, Paget's disease, hypercalcemia, bone metastasis of cancer andosteoporosis. In addition, their pharmacological actions, for example,prevention of aggravation after cancer metastasis, relief of bone pain,prevention of periodontal diseases, etc. are known.

Furthermore, due to the affinity with calcified tissues, the compound ofthe present invention can be used in diagnosis and medical treatment ofnot only bone diseases but also calcified lesions of blood vessels, suchas arteriosclerosis at calcified blood vessel sites or the like, andchronic inflammatory diseases.

Accordingly, since the compound of the present invention selectivelyaccumulates in calcified tissues in general in a living body and israpidly excreted in urine, it is useful as a diagnostic agent or atherapeutic agent of various diseases mentioned above.

Specifically, the compound of the present invention suitably subjectedto radioactive nuclide labeling is useful as an active ingredient fordiagnosing bone diseases such as bone metastasis, osteoporosis, Paget'sdisease, bone fracture, heterotopic ossification, bone formation or bonedissolution and for diagnosing calcified blood vessel sites includingarteriosclerosis. When it is applied to visualization of the skeletalscintigraphy for finding out affected sites, the compound of the presentinvention is intravenously administered to a body of mammals includinghumans, and, subsequently radioactivity distribution in the body ismeasured. The radioactivity distribution is measured by using a commonlyknown equipment (gamma camera, etc.).

In addition, the compound of the present invention is applicable astherapeutic agents for inflammatory bone diseases such as rheumatoidarthritis and lumbago and for pain relieving, and as anticancer drugsfor bone cancer and its metastasis, prevention of bone metastasis ofcancer, and the like. Furthermore, the compound of the present inventioncan be used in diagnosis for selection of a therapeutic agent and alsofor pharmacometrics such as efficacy estimation.

The compound of the present invention can be used as an imaging agent invarious types of diagnostic imaging using radiation, nuclear magneticresonance, X-ray, ultrasonic wave, etc., or a therapeutic agentdepending on the type of labeling substance contained therein. Thecompound of the present invention or a salt thereof can be provided as apharmaceutical composition with at least one pharmaceutically acceptablecarrier.

BEST MODE FOR CARRYING OUT THE INVENTION

(1) Mother Nucleus MC

In the present invention, the mother nucleus MC is not limited as longas it has a plurality of functional groups available for chemicallybonding to the group AC having affinity with a calcified tissue and theligand LI. Specifically, a residue of a compound is included which has aplurality of functional groups selected from the group consisting of anamino group, an amide group, a hydroxyl group, a thiol group, athioether group, a sulfonyl group, a phosphonyl group, an aldehydegroup, a carboxyl group, a carbonyl group, a halogen and a cyano group.

Therefore, the mother nucleus MC may be a monosaccharide,oligosaccharide, polysaccharide, amino acid, oligopeptide, polypeptide,nucleotide, oligonucleotide, poly nucleotide, protein, protein fragment,chemical derivatives thereof, or a synthetic polymer, as long as it hasa plurality of the above-mentioned functional groups.

The molecule size of the compound of the present invention can becontrolled by the size of the mother nucleus MC, and therefore, transferof the compound between blood vessel and tissues can be controlled bychanging the molecule size thereof depending on the specific use so thatthe compound can effectively pass through capillary vessel pores (5 to10 μm) and selectively act on the target tissue.

Preferably, the mother nucleus MC is a residue of a saccharide compoundselected from the group consisting of a monosaccharide, anoligosaccharide, an amino oligosaccharide, a cyclodextrin and asaccharide dendrimer, more preferably, a residue of an oligosaccharide,an amino oligosaccharide, a cyclodextrin, a saccharide dendrimer, andparticularly preferably a residue of an oligosaccharide and an aminooligosaccharide.

The monosaccharide includes glucose, mannose, galactose, glucosamine,mannosamine, galactosamine, etc.

The oligosaccharide includes those containing as a constituentmonosaccharide one or more selected from the group consisting ofglucose, mannose, galactose, etc. These oligosaccharides may be linearor branched, and it is preferable from a viewpoint of thetransferability of the compound of the present invention between theblood vessel and tissues that the oligosaccharide is a polymer of 2 to20 saccharide units. The oligosaccharide may be those formed ofconstituent monosaccharides that are mutually α- or β-linked, or thoseformed of constituent monosaccharides that are mutually 1-3,1-4 or1-6-linked. Specific examples of preferable oligosaccharides includemaltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose,isomaltotriose, isomaltotetraose, isomaltopentaose, isomaltohexaose,isomaltoheptaose, cellotriose, cellotetraose, cellopentaose,cellohexaose, laminaritriose, laminaritetraose, laminaripentaose,laminarihexaose, laminariheptaose, erlose, panose, raffinose, etc. Theoligosaccharide may be those reduced in some parts such as at a terminalend or those not reduced, but those reduced are preferable.Dialdehydesaccharides thereof are also included in the oligosaccharide.

The amino oligosaccharide includes those containing as a constituentmonosaccharide one or more aminosaccharide selected from the groupconsisting of glucosamine, mannosamine, galactosamine, etc. These may belinear or branched, and it is preferable from a viewpoint of thetransferability of the compound of the present invention between theblood vessel and tissues that the amino oligosaccharide is a polymer of2 to 20 saccharide units and a polymer of 2 to 13 saccharide units ismore preferable. The amino oligosaccharide may be those formed ofconstituent monosaccharides that are mutually α- or β-linked, or thoseformed of constituent monosaccharides that are mutually 1-3,1-4 or1-6-linked. Chitosan oligosaccharides having 2 to 10 repeting units ofconstituent monosaccharide or galactosamine oligosaccharides having 2 to10 repeting units of constituent monosaccharide can be used asparticularly preferable amino oligosaccharides, and specificallychitosan oligosaccharides such as chitosan dimer, chitosan trimer,chitosan tetramer, chitosan pentamer and chitosan hexamer andgalactosamine oligosaccharides such as galactosamine dimer,galactosamine trimer, galactosamine tetramer, galactosamine pentamer andgalactosamine hexamer can be exemplified. The amino oligosaccharide maybe those reduced in some parts such as at a terminal end or those notreduced, but those reduced are preferable. In addition, aminooligosaccharides in which a part of the amino groups is N-acetylated anddialdehyde saccharides thereof are also included in the aminooligosaccharide.

The cyclodextrin includes α-, β- and γ-cyclodextrins. Dialdehydesaccharides in which the positions 2 and 3 of the constituentmonosaccharide are reduced are also included in the cyclodextrin.

The saccharide dendrimer includes, for example, a linear or branchedsaccharide bonded to a core consisting of a polycarboxylic acid or alkylpolycarboxylic acid. The structure of the saccharide dendrimer isexpressed by generation which means a structure where circles can bedrawn from the inner core to the outside, and the number of the circlesof polycarboxylic acid is preferably 1 to 5 generations, more preferably1 to 3.

Another saccharide dendrimer includes a linear or branched saccharidebonded to a core consisting of a polyamine or alkylpolyamine. Thissaccharide dendrimer takes a structure where circles can be drawn fromthe inner core to the outside on the basis of the constituting nitrogenatoms, and saccharides are bonded to the outermost nitrogen atoms toconstitute the saccharide dendrimer. In this case, the number of thenitrogen circles is preferably 1 to 5 generations, more preferably 1 to3.

In addition, the mother nucleus MC which has a phosphonyl group includesinositol trisphosphates, etc., and the mother nucleus MC which has asulfonyl group includes chondroitin sulfates, heparan sulfates, keratansulfates, etc. The mother nucleus MC which has a carboxyl group or acarbonyl group includes glucuronic acid, etc., and the mother nucleus MCwhich has a halogen includes acetobromo-α-D-glucuronic acid methylester,etc., and the mother nucleus MC which has a cyano group includescyanomethylmannose, etc.

(2) Group AC Having Affinity with a Calcified Tissue

The group AC having affinity with a calcified tissue is not restrictedas long as the compound has affinity with a calcified tissue found inbones, blood vessels, etc., and typically includes polyaspartic acid,polyglutamic acid, organic phosphonic acid and derivatives thereof.

The organic phosphonic acid which constitutes the group AC havingaffinity with a calcified tissue includes, for example, a residue of adiphosphonic acid represented by the following formula I, derivativesthereof, and salts thereof.(wherein,

R¹ and R³, which are the same or different, each represents a formula—(CR⁵R⁶)_(k)—R⁷, —(CR⁸R⁹)_(m)—R¹⁰ _(n)—(CR¹¹R¹²)_(o)—R¹³_(p)—(CR¹⁴R¹⁵)_(q)R¹⁶ (wherein R⁵, R⁶, R⁸, R⁹, R¹¹, R¹², R¹⁴, R¹⁵ andR¹⁶ are groups each independently selected from the group consisting ofH, —OH, —COOH, —C(NH₂)═NH, —CN, —SO₃H, —NR¹⁷ ₂ and a halogen atom, R¹⁷is independently H or —(CH₂)_(r)CH₃ respectively, R⁷, R¹⁰ and R¹³ aregroups each independently selected from the group consisting of sulfur,oxygen, amide, imide, a divalent heterocycle consisting of 3 to 12 atomsand a cyclic hydrocarbon (Ar—(R¹⁸ _(r)—R¹⁹)_(s)), R¹⁸ is —CR⁵R¹⁷, R¹⁹ isindependently selected from the group consisting of H, —OH, —COOH,—C(NH₂)═NH, —CN, —SO₃H, —NH₂, —NHMe, —NMe₂ and a halogen atom; k, l, m,n, o, p and q are each independently 0 or an integer of 1 or more, r is0 to 3, s is 0 to 12, and the sum total of k, l, m, n, o, p and q is 0to 12);R² is a group selected from H, —OH, —NH₂, —NHMe, —NMe₂, —CN, and a loweralkyl group (which may be substituted with one or a plurality of polargroups);R⁴ is a group selected from H, —OH, —NH₂, —NHMe, —NMe₂, —CN, —SO₃H, ahalogen and a lower alkyl group (which may be substituted with one or aplurality of polar groups); andj is 0 or 1 (provided that, when j is 0, R¹ is not be H and when j is 1,both of R¹ and R³ cannot be H).

The organic phosphonic acid represented by the above-mentioned formula Iincludes, for example, ethylene glycol-1,2-bisphosphonic acid,diphosphonomethanesulfonic acid, 2,2-diphosphonoethane-sulfonic acid,2,2-diphosphono-2-hydroxyethane-sulfonic acid,1,1-diphosphono-2-hydroxyethane-sulfonic acid,N,N-dimethyl-1-aminoethane-1,1-diphosphonic acid, the derivativesthereof, etc.

As an organic phosphonic acid represented by the above-mentioned formulaI, α-geminal-bisphosphonic acid, i.e., the bisphosphonic acid in which jis 0 and which has P—C—P bond or derivatives thereof can be usedpreferably. In this case, R¹ and R² which are the substituent groups onthe alpha carbon may be hydrogen, a hydroxyl group, an amino group, ahalogen group, a carboxylic acid group, a sulfonic acid group, a loweralkyl group, a lower alkyl alcoholic group, a cyano group, etc., andeither R¹ or R² bonds to the functional group of the mother nucleus MC.

That is, among the compounds represented by the above-mentioned formulaI, compounds in which j is 0 (therefore, R¹ is not H) and R¹ isrepresented by the formula (CR¹⁴R¹⁵)_(q)R¹⁶ (wherein R¹⁴, R¹⁵ and R¹⁶are groups each independently selected from the group consisting of H,—OH, —COOH, —C(NH₂)═NH, —CN, —SO₃H, —NR¹⁷ ₂ and a halogen atom; R¹⁷ isindependently H or —(CH₂)_(r)CH₃; respectively; q is 0 or an integer of1 to 9; and r is 0 to 3) or the formula —R¹³p-R¹⁶ (wherein R¹⁶ is agroup selected from the group consisting of H, —OH, —COOH, —C(NH₂)═NH,—CN, —SO₃H, —NR¹⁷ ₂ and a halogen atom; R¹⁷ is independently H or—(CH₂)_(r)CH₃, r is 0 to 3; R¹³ is a group represented by thiophenylene,pyrimidinylene, pyrrolidinylene, phenylene, ether, or thioether) and R²is H or —OH are preferable.

Among the compounds represented by the above-mentioned formula I,compounds in which j is 0 (therefore, R¹ is not H) and R¹ is representedby the formula —(CR¹⁴R¹⁵)_(q)R¹⁶ (wherein R¹⁴ and R¹⁵ are H, R¹⁶ is agroup each independently selected from the group consisting of H, —OH,—COOH, —C(NH₂)═NH, —CN, —SO₃H, —NR¹⁷ ₂ and a halogen atom; R¹⁷ isindependently H or —(CH₂)_(r)CH₃; r is 0 to 3; q is 0 or an integer of 1to 9, preferably 0 or an integer of 1 to 5) and R² is H or —OH are morepreferable.

Such bisphosphonic acids include, for example, methane diphosphonic acid(MDP), hydroxymethane diphosphonic acid (HMDP),1-hydroxyethane-1,1-bisphosphonic acid (EHDP), dimethylamino methylenediphosphonic acid (DMAD), 3,3-diphosphono-1,2-propane diphosphonic acid(DPD), Tildronate, etc., and an ester or salt thereof can also be used.The structural formulae of these compounds are as follows:

Methane diphosphonic acid (MDP) etc. can be used as an organicphosphonic acid of the present invention by deriving as shown belowaccording to a known method (for example, a method described in J. Org.Chem., 66 (11), 3704-3708, (2001)).

Alendronate which is a hydroxybisphosphonic acid can be used as anorganic phosphonic acid of the present invention according to a knownmethod (for example, a method described in Heteroatom Chemistry, 11 (7),556-561 (2000)) as shown below.

The method of chemically bonding the above-mentioned organic phosphonicacid to the mother nucleus MC includes amidation, esterification,imidation, etc., for example.

Furthermore, as organic phosphonic acids, organic aminophosphonic acidderivatives in which the group represented by the formula II is bondedto an amine nitrogen atom, an ester or salt thereof can also be used.

(wherein t is an integer of 1 to 8; X and Y are each independentlyselected from hydrogen, a halogen group, a hydroxyl group, a carboxylgroup, a carbonyl group, a phosphonic acid group, and a hydrocarbongroup having 1 to 8 carbon atoms, and when t is larger than 1, each Xand Y may be the same or different; R²⁰ is selected from hydrogen, asilyl group, an alkyl group, a benzyl group, sodium and potassium).

Another example of the organic phosphonic acid includes phosphonic acidderivatives represented by the formula III, esters or salts thereof.

(wherein each u and u′ is independently an integer of 0 to 5, andpreferably is 0, 1, or 2; R²¹, R²² and R²³ are each independently—(CH₂)_(v)— (v=1 to 5); A, B, C, D, E, and F are each independentlyselected from the group consisting of hydrogen, a methyl group, an ethylgroup, an isopropyl group, a pivaloyl group, a benzyl group, an acetylgroup, a trifluoroacetyl group, and groups of the following formulaeIV-1 to 3, and one of A, B, C, D, E and F is the group of followingformula IV-1.

(wherein t, X and Y are the same as in the above-mentioned formula II;t′ is 2 or 3; X′ and Y′ are each independently selected from hydrogen, amethyl group and an ethyl group, and each X′ and Y′ may be the same ordifferent)).

Examples of the phosphonic acid derivative represented by the formulaIII include ethylenediaminetetramethyleneprosphonic acid (EDTMP),diethylenetriaminepentamethylenephosphonic acid (DTPMP),hydroxyethylethylenediaminetrimethylenephosphonic acid (HEEDTMP),nitrilotrimethylenephosphonic acid (NTMP), tris(2-aminoethyl)aminehexamethylenephosphonic acid (TTHMP),1-carboxyethylenediaminetetramethylenephosphonic acid (CEDTMP),bis(aminoethylpiperazine) tetramethylenephosphonic acid (AEPTMP),N-methylethylenediaminetrimethylenephosphonic acid (MEDTMP),N-isopropylethylenediaminetrimethylenephosphonic acid (IEDTMP),N-benzylethylenediaminetrimethylenephosphonic acid (BzEDTMP), etc.

Other organic phosphonic acids which can be used include polyvalentphosphoric acid derivatives typically exemplified by EDTMP, DTPMP, etc.Similarly to bisphosphonic acids, these compounds have affinity withcalcified tissues such as bone, and are considered promising astherapeutic agents.

Among the polyaspartic acids, those of polymerization degree of 4 to 12can be used preferably. Among the polyglutamic acids, those ofpolymerization degree of 4 to 12 can be used preferably.

The method of chemically bonding the above-mentioned organic phosphonicacid, polyaspartic acid, or polyglutamic acid to the mother nucleus MCincludes amidation, esterification, imidation, etc., for example.

(3) Ligand (LI)

The ligand (LI) which is capable of binding to a metal atom in thecompound of the present invention includes, for example, the ligandwhich can form a complex with a metal atom or a metal ion. The ligand(LI) as used herein means a compound which can form a stable complexwith a metal atom or a metal ion.

Typical examples of the ligand (LI) include polydentate or multidentateligands i.e., ligands which contain a plurality of coordinating atomsper one ligand molecule. The coordinating atom as used herein is definedas an atom having a free electron pair which can bind to a metal atom.This molecule has preferably a plurality of coordinating atoms. Thecoordinating atom is selected from nitrogen, oxygen, sulfur, phosphorusand carbon, and nitrogen and/or oxygen and/or sulfur are suitablecoordinating atoms.

Typical examples of the polydentate or multidentate ligand include chainor cyclic polyaminopolycarboxylic acid or chain or cyclicpolyaminopolyphosphonic acid, or derivatives thereof. Specific examplesof the polyaminopolycarboxylic acid include ethylenediaminediaceticacid, nitrilotriacetic acid, ethylenediaminetetraacetic acid(hereinafter abbreviated as EDTA), diaminocyclohexanetetraacetic acid,diethylenetriaminepentaacetic acid (DTPA),triethylenetetraminehexaacetic acid (TTHA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),1,4,8,11-tetraazacyclotetradecane-1, 4,8,11-tetraacetic acid (TETA) andderivatives thereof. Specific example of polyaminopolyphosphonic acidincludes EDTMP and derivatives thereof.

The derivatives of polyaminopolycarboxylic acids include, for example,compounds in which one or a plurality of carboxyl groups ofpolyaminopolycarboxylic acids are subjected to esterification,halogenation, addition of protection group, or replacement with ahydrocarbon group which has a substituent group other than carboxylicacid; compounds in which a hydrocarbon group having a substituent groupother than carboxylic acid or a substituent group having no hydrocarbongroup is introduced into a hydrocarbon moiety which constitutes thepolyaminopolycarboxylic acid; and compounds in which an ether group etc.is introduced into a carbon backbone moiety of thepolyaminopolycarboxylic acid. Specifically, hydroxyethylethylenediaminetriacetic acid, diaminopropanoltetraacetic acid,N,N-bis(2-hydroxybenzyl) ethylenediaminediacetic acid, glycol etherdiaminetetraacetic acid, etc. can be mentioned.

In addition to these, those selected from the group consisting ofcyclam, N{1-2,3-dioleyloxy)propyl}-N,N,N-triethylammonium (DOTMA),mercaptoacetylglycylglycylglycine (MAG3), ethylene cysteine dimer (ECD),hydrazinonicotinyl (HYNIC), lysine-tyrosine-cysteine (KYC),cysteine-glycine-cysteine (CYC),N,N′-bis(mercaptoacetamido)ethylenediamine (DADS),N,N′-bis(mercaptoacetamido)-2,3-diamine propanoic acid (CO₂DADS)(European Patent No. 0173424, U.S. Pat. No. 4,673,562 specification),N,N′-bis(2-mercaptoethyl)ethylenediamine (BATs) (European Patent No.0163119 and No. 0200211), thiosemicarbazone, propylene amineoxime(PnAO), other amineoxime ligand (European Patent No. 0123504 and No.0194843) and derivatives thereof can be exemplified as ligand (LI).

In addition to these, as a ligand (LI), a group of multidentate ligandcontaining sulfur and nitrogen as coordinating atoms such as6-hydrazinonicotinic acid, diaminodithiol, monoaminomonoamidodithiol,diamidodithiol and triamidothiol can be also used for a complexformation part. Referring to chelating groups, for example,diaminodithiol includes N,N′-bis(2-mercaptoethyl)ethylenediamine,2,2,9,9-tetramethyl-4,7-diaza-1,10-decanethiol;monoamidemonoaminodithiol includesN-2-mercaptoethyl-2-mercaptoethylaminoacetamide andN-(2-mercaptoethyl)aminoethyl-2-mercaptoacetamide; diamidodithiolincludes 1,2-ethylene bis(2-mercaptoacetamide); and triamidothiolincludes mercaptoacetylglycylglycylglycine.

The multidentate ligand further includes huge 4-, 5-, 6-, 7- and8-dentate compounds containing nitrogen which are cyclic or open-chainand may or may not have other coordinating atoms or unsaturated bonds.

Bonding of these ligands (LI) and the mother nucleus MC can be performedby chemically bonding functional groups of the ligands which are notimportant for forming a complex with metal and the functional group ofthe mother nucleus MC mutually.

The ligand (LI) may be a bifunctional ligand. The bifunctional ligand isa compound that has in molecule a site for binding to the mother nucleusMC and a site for forming a complex with a metal. Therefore, by way ofthe functional groups which exist in the mother nucleus MC and areavailable for binding to the bifunctional ligand, the bifunctionalligands to the number corresponding to the number of the functionalgroups can be chemically bonded to the physiologically acceptablesubstances.

The site for forming a complex with a metal is not limited to specifictypes as long as it is a multidentate ligand which forms a stablecomplex with a respective metal, and it can be typically selected fromcyclic or chain polyaminopolycarboxylic acid or cyclic or chainpolyaminopolyphosphonic acid as mentioned above and, for example, EDTA,DTPA, DOTA, TETA or derivatives thereof, or EDTMP or derivativesthereof, or MAG3, cyclam, BAT, ECD, DADS and PnAO or derivatives thereofcan be used.

As a reactive coupling group in the bifunctional ligand whichconstitutes a site for binding to the mother nucleus MC, not onlynormally-employed amino group, carboxyl group and thiol group, but alsoactive halogen, alkoxy ester, N-hydroxysuccinimide ester, imide ester,maleimide, thiophthalimide, isothiocyanate, acid anhydride, etc. can bespecifically exemplified.

Specific examples of the bifunctional ligand include compounds havingpolyaminopolycarboxylic acid or polyaminopolyphosphonic acid as a sitefor forming a complex with a metal, for example,1-(p-isothiocyanatebenzyl)-DTPA [Martin W B et al., Inorg. Chem., 25, p.2772-2781 (1986)], anhydrous DTPA,2-(p-isothiocyanatebenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid [U.S. Pat. No. 4,678,667 specification],succinimidyl-6-hydrazinonicotinate [Abrams, M. J. et al., J. Nucl. Med.,31, p. 2022-2028 (1990)], DTPA-mono(2-aminoethylamide),DTPA-mono(3-aminopropylamide), DTPA-mono(6-aminohexylamide) [JapanesePatent No. 2815615], 1-(4-aminobenzyl)-EDTA,1-(4-isothiocyanobenzyl)-EDTA, 1-[4-(3-maleimidepropyl)amidebenzyl-EDTA, 1-[4-(5-maleimidepentyl)amidobenzyl]-EDTA, etc.

The bonding of the mother nucleus MC and a bifunctional ligand can beperformed by a method known per se. For example, the reaction of themother nucleus MC and a bifunctional ligand having, as a reactivecoupling group, acid anhydride [Hnatowich D J et al., Int. J. Appl. Rad.Isot., 33, p. 327-332 (1982)], isothiocyanate [Esteban J M et al., J.Nucl. Med., 28, p. 861-870 (1987)], alkoxyester [Washburn LC et al.,Nucl. Med. Biol., 18, p. 313-321(1991)] or active halogen [Fourie P J etal., Eur. J. Nucl. Med., 4, p. 445-448 (1979)] can be performedaccording to the description of the known literatures cited above.

A preferable ligand (LI) is selected from the group consisting ofethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), triethylenetetraminehexaacetic acid (TTHA), cyclam,1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),N{1-2,3-dioleyloxy)propyl}-N,N,N-triethylammonium (DOTMA),mercaptoacetylglycylglycine (MAG3), ethylene cysteine dimer (ECD),hydrazinonicotinyl (HYNIC), lysine-tyrosine-cysteine (KYC),cysteine-glycine-cysteine (CYC),N,N′-bis(mercaptoacetamido)ethylenediamine (DADS),N,N′-bis(mercaptoacetamido)-2,3-diamine propanoic acid (CO2DADS),N,N′-bis(2-mercaptoethyl)ethylenediamine (BATS), thiosemicarbazone,propylene amineoxime (PnAO), and other amineoxime ligand and derivativesthereof.

Complexation of the metal atom to the above-mentioned ligand (LI) can beperformed by ordinary methods. The metal atom for complexation can besuitably selected according to use, and a metal atom useful as alabeling substance can be usually selected. The metal atom includes ametal atom having radioactivity, paramagnetism or X-ray impermeabilityor an ion thereof.

The compound of the present invention can include a metal atom or anisotope of halogen atom, carbon, oxygen, nitrogen, sulfur or phosphorusin at least one of the mother nucleus MC and the group AC havingaffinity with a calcified tissue, irrespective of the existence ofligand LI. As a halogen atom, fluorine, bromine, iodine, etc. can beused preferably. Introduction of these halogen atoms can be performed byintroducing a substituent group containing a leaving group such as atosyl group into the mother nucleus MC or the group AC having affinitywith a calcified tissue, and replacing this substituent group with ahalogen atom. It can be also performed by introducing a substituentgroup such as trialkyl tin represented by the formula Sn(R₃) and othermetal alkyl groups into the mother nucleus MC or the group AC havingaffinity with a calcified tissue, and replacing this substituent groupwith a halogen atom. A methyl group, an ethyl group, a propyl group, abutyl group, etc. can be used as the alkyl group of the metal alkylgroup. A preferable metal alkyl group is trimethyltin or tributyltin.

Alternatively, the isotope elements, for example, 11-C can be introducedby reacting methyl iodide (¹¹CH₃I) with the amino group of glucosamineof the mother nucleus MC (yielding —NH¹¹CH₃) or by reacting a carboxylicacid of polycarboxylic acid (DTPA etc.) which constitutes theabove-mentioned ligand (LI) with a methylamine (¹¹CH₃NH₂) (yielding—CONH¹¹CH₃). Moreover, they can be also introduced by using iodineglucose derivatives (see Japanese Patent Laid-Open No. 09-176179,Japanese Patent Laid-Open No. 09-176050 and Japanese Patent Laid-OpenNo. 07-267980).

The metal atom which constitutes the complex mentioned above or themetal atom or isotope element contained in the mother nucleus MC, thegroup AC having affinity with a calcified tissue or the ligand LI issuitably selected according to use.

For use as radioactive diagnostic agents, those which emit gamma ray butdo not remarkably injure underlying normal organs or tissues afteradministration are selected. As radioactive nuclides, those which havegamma ray capable of imaging or those which can be mixed (doped) with aradioactive nuclide that contains gamma ray capable of imaging arepreferable. This doping radioactive nuclide may be the same or adifferent element as long as its chemical properties are sufficientlyclose to those of beta emitter nuclides and its distribution in theliving body according to the present invention is the same as or closeto the distribution of the beta emitter.

For use as radioactive therapeutic agents, those which emit betaparticles but, after administration, do not remarkably injuringunderlying normal organs or tissues while treating affected regions areselected. These radioactive nuclides have an average β energy of 0.25 to2.75 Mev, but do not need to have gamma ray that enables imaging, andmay suitably have an average soft tissue penetration degree of 0.70 to25.0 mm and a half-life of 0.05 to 700 hours.

Preferable metal atoms and isotope elements include elements selectedfrom the group consisting of the elements of atomic number 6-9, 15-17,21-29, 31, 35, 37-44, 49, 50, 53, 56-70, 72-75, 81, 83 and 85. Similarlypreferable metal atoms and isotope elements include radioactive nuclidesselected from the group consisting of 11-C, 15-0,18-F, 32-P, 59-Fe,67-Cu, 67-Ga, 81-Rb, 89-Sr, 90-Y, 99m-Tc, 111-In, 123-I, 124-I, 125-I,131-I, 117m-Sn, 153-Sm, 186-Re, 188-Re, 201-Tl, 211-At, 212-Bi and213-Bi, and among these, preferred are radioactive nuclides selectedfrom the group consisting of 32-P, 59-Fe, 67-Cu, 67-Ga, 81-Rb, 89-Sr,90-Y, 99m-Tc, 111-In, 123-I, 124-I, 125-I, 131-I, 117m-Sn, 153-Sm,186-Re, 188-Re, 201-Tl and 212-Bi.

When the compound of the present invention is used for a nuclearmagnetic resonance (MRI) diagnostic agent, preferable metal atoms andisotope elements include elements selected from the group consisting ofchromium (III), manganese (II), iron (II), iron (III), praseodymium(III), neodymium (III), samarium (III), ytterbium (III), gadolinium(III), terbium(III), dysprosium (III), holmium (III), and erbium (III).

When the compound of the present invention is used for X-ray orultrasonic diagnosis, preferable metal atoms and isotope elementsincludes elements selected from the group consisting of bismuth,tungsten, tantalum, hafnium, lantern, lanthanide, barium, molybdenum,niobium, zirconium and strontium.

(4) Linker

The above-mentioned group AC having affinity with a calcified tissue andthe ligand LI may be coupled with the mother nucleus AC by way of alinker L. As such a linker L, can be used polylysine, other peptides,alkyl, polyacrolein, and alkyl ethers, alkylamide, alkylamine and analkylolefin represented by formula —(CH₂)_(w)—R²⁴—(CH₂)_(w)— (wherein wis each independently 0 to 5, and R²⁴ is O, S, NHCO, NH, or CH═CH), aswell as divalent reagents that are used in enzyme immunoassay, etc.

The divalent reagents include, for example,N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),ethyleneglycol-O,O′-bis(succinimidylsuccinate) (EGS),N-(4-maleimidebutyryloxy)succinimide (GMBS),N-(4-maleimidebutyryloxy)sulfosuccinimide sodium salt (Sulfo-GMBS),N-(6-maleimidecaproyloxy)sulfosuccinimide sodium salt (Sulfo-EMCS),N-(8-maleimidecapryloxy)sulfosuccinimide sodium salt (Sulfo-HMCS),N-(11-maleimideundecanoyloxy)sulfosuccinimide sodium salt (Sulfo-KMUS),3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP), glutaraldehyde,etc.

The reaction of the linker L and the mother nucleus MC, and the reactionof the linker L, the above-mentioned ingredient AC and the ligand LI canbe carried out by methods known per se. For example, the reaction forcoupling DTPA-mono(2-aminoethylamide) or DTPA-mono(6-aminohexylamide)with an amino group of IgG or Fab fragment through EGS or DTSSP can beperformed according to the method of Japanese Patent No. 2815615. Incase of using polylysine, the reaction can be performed according to themethod of Japanese Patent No. 2548711, and in case of usingpolyacrolein, the reaction can be performed according to the method ofJapanese Patent No. 1729192, and when dialdehyde starch and aminooligosaccharide are used as the mother nucleus MC, the reaction can beperformed according to Japanese Patent No. 1721409, Japanese PatentLaid-Open No. 7-206895, etc.

(5) Preferable Compounds

According to preferable embodiment of the present invention, compoundsrepresented by the following general formula V-1 or V-2 are provided.

(wherein R and R′ are each independently an group AC having affinitywith a calcified tissue or a ligand LI for binding to a metal atom, andat least one of them is the group AC having affinity with a calcifiedtissue; x and y are each independently 0 to 19; and x+y is 1 to 19).

The compound represented by the above-mentioned general formula can beeasily obtained by reacting a bisphosphonate compound (BP) as a group AChaving affinity with a calcified tissue to an amino group of theaminooligosaccharide consisting of one or more kinds of monosaccharidesselected from the group consisting of glucosamine, mannosamine andgalactosamine which constitute the mother nucleus MC, and reacting thecarboxylic acid group of polyaminopolycarboxylic acid as the ligand LI.In this case, the terminal end of the aminooligosaccharide may bereduced but does not need to be reduced. However, when the terminal endis reduced, the amino group at the terminal end of theaminooligosaccharide can be selectively modified with a protectinggroup, and consequently a desired compound can be chemically bonded tothe amino group at the terminal end of the reduced oligosaccharide andtherefore it is convenient. When this reaction is performed, the reducedamino oligosaccharide has preferably 2 to 50 saccharide units, morepreferably 2 to 20 saccharide units, and particularly preferably 2 to 13saccharide units.

In this way, according to another aspect of the present invention, amethod of selectively modifying an amino group at a terminal end isprovided which comprises providing an amino oligosaccharide having 2 to50 saccharide units which consists of one or more monosaccharidesselected from the group consisting of glucosamine, mannosamine andgalactosamine and is reduced at a terminal end thereof, and subjectingthe amino oligosaccharide to a reaction for generating a carbamatecompound.

In addition, according to still another aspect of the present invention,a method of selectively modifying an amino group at a terminal end witha butoxycarbonyl (Boc) group is provided which comprises reacting, withdibutyl dicarbonate, an aminosaccharide of 2 to 13 saccharide unitswhich consists of one or more monosaccharides selected from the groupconsisting of glucosamine, mannosamine and galactosamine and is reducedat a terminal end thereof.

The above-mentioned modifying method is convenient for an intermediateof the compound represented by the formula (AC)_(a)-MC which is acompound having affinity with a calcified tissue according to thepresent invention.

The most preferable compound of the present invention can be representedby the following chemical formulae.

(6) Formulation, Kit and Dosage

The compound of the present invention may be in a form of salt, hydrate,and solvate. As the salt, mention may be made of pharmaceuticallyacceptable salts with inorganic bases such as salts with alkaline metalssuch as lithium, sodium and potassium, salts with alkaline-earth metalssuch as salts with calcium and magnesium, ammonium salts, salts withorganic bases such as methylamine, ethylamine, dimethylamine,diethylamine, trimethylamine, triethylamine, cyclohexylamine,ethanolamine, diethanolamine, morpholine and meglumine, salts with basicamino acids such as lysine, ornithine and arginine. Preferably sodiumand potassium can be suitably used.

The compound of the present invention can be used in a form of anaggregate, an aqueous solution or a lyophilized product. For example, itcan be in a form of a kit for preparing a radioactive labeled compoundby allowing a lyophilized preparation to co-exist with a reducing agent,a stabilizing agent, etc. The kit for preparing a radioactive labeledcompound containing the compound of the present invention is preferablyprovided as a lyophilized preparation which is dissolved in a suitabledilution agent when it is used, and which is labeled with a radioactivenuclide such as technetium or rhenium metal salt upon administration.Alternatively, it may be in a form of the above-mentioned aqueoussolution agent, and labeled with a radioactive transition metal such astechnetium or rhenium metal salt by commonly-used drug preparationtechniques or in a presence of a non-metallic reducing agent.

As for forms of the compound of the present invention, a form in whichthe mother nucleus MC or the group AC having affinity with a calcifiedtissue is substituted with a substituent group which contains a halogengroup, a leaving group or a metal alkyl group is convenient. The halogenatom is preferably fluorine, bromine, iodine, etc., and the metal alkylgroup includes trialkyl tin represented by the formula Sn(R₃), etc., andthe alkyl group includes a methyl group, an ethyl group, a propyl group,a butyl group, etc. Preferably, trimethyltin or tributyltin is be used.The labeling reaction of the substituent group X with a radioactivehalogen is performed by known methods, preferably performed by asubstitution reaction or exchange reaction.

Conventionally used additives such as an oxidizing agent, stabilizingagent, buffering agent and excipient may be added to the kit forpreparing the radioactive compound. For example, chloramine T orhydrogen peroxide as needed is added as an oxidizing agent, followed bythe labeling reaction. This radioactive halogen labeling may beperformed by a known method, in which temperature, concentration, pH,etc. are not particularly limited.

As a reducing agent used for chemical reduction of peracid ions such as99m-pertechnetate (99 mTcO₄ ⁻) in performing labeling a radioactivetransition metal, can be generally used metal ions such as of tin, zincand iron, or metallic compounds such as chromium chloride and chromiumacetate, including tin chloride, stannous fluoride, etc., butnonmetallic reducing agents such as sodiumdiphenylphosphinobenzene-3-sulfonate, formamidinesulfonic acid orglucoheptanoic acid can be also used besides the metallic compounds. Itis also possible to use dithionic acid and sodium bisulfite. Inaddition, by using compounds which form a relatively unstable complexand are exemplified by organic acid such as gluconic acid, ascorbic acidand citric acid and a saccharide such as mannose, it is possible toperform a complex exchange reaction with the compound of the presentinvention and to transfer the radioactive transition metal, therebyperforming the labeling. There is no particularly restriction on thereaction conditions such as temperature, concentration, and pH, andusually the reaction can be performed at room temperature or underheating, and a reducing agent is suitably used according to the reactionconditions of these reactions.

When a physiological saline which contains pertechnetate eluted from a99 mTc generator is added to a preparation of the kit according to thepresent invention that contains a reducing agent, technetium is reducedfrom heptavalence to the lower number of oxidization by the reducingagent and forms a complex with DTPA. Although the exact character of theformed complex is unknown, it would be possible that the reducing agentcan also form a part of these complexes. However, since the compoundrepresented by the formula (AC)_(a)-MC-(LI)_(b) of the present inventionhas a ligand LI, it is possible to obtain a complex in which thecomponent AC does not participate in complexation due to the differencein coordinating ability between the ligand LI and the component AC.After the liquid is shaken, heated or allowed to stand still in order toensure completion of reduction and formation of a complex, the liquidcan be used for injection.

The compound of the present invention may contain physiologicallyacceptable buffering agents (for example, pH adjusting agents such as aphysiological saline, acetic acid, phosphoric acid, carbonic acid andtris(hydroxymethyl)aminomethane, etc.) and other physiologicalacceptable additives (for example, stabilizing agents such as ascorbicacid and paraben, dissolution agents such as meglumine and betaine, andexcipients such as D-mannitol).

The compound of the present invention can be used in a similar manner toconventional diagnostic agents or therapeutic agents, and, for example,it can be used as a liquid preparation administered to human and othermammals by injection. The dose is substantially the same as that ofconventional diagnostic agents or therapeutic agents, and about 3 to 25MBq/kg, preferably 6 to 12 MBq/kg for diagnostic agents, and depends onradioactive nuclides for therapeutic agents. The dose is suitably varieddepending on kinds of compounds, kinds of radioactive nuclides to beused, age, weight, condition of patients, administration methods, andother agents used in combination, and the like.

EXAMPLES

Below, the present invention will be further described in detail by wayof Examples, but the present invention is not limited to these Examples.Analysis was performed using methods well-known to those skilled in theart. In the Examples, measurement of NMR spectrum was performed usingJNM-500 (manufactured by JEOL) and measurement of MS spectrum wasperformed using Q-Tof type (manufactured by Micromass). Theabbreviations used in Examples are as follows:

WSCD: 1-ethyl-3-(3-diethylaminopropyl)carbodiimide,

HOBt-H₂O: 1-hydroxytriazole monohydrate,

DTPA: diethylenetriaminepentaacetic acid,

HPLC: High Performance Liquid Chromatography,

—OAc: acetyl group,

Boc-: tert-butoxycarbonyl group,

Bn-: benzyl group.

The “desalting” described below was performed under the followingconditions:

Electric dialysis equipment: MICROACILYZER G3 (trade name, manufacturedby Asahi Chemical Co., Ltd.);

Dialysis membrane: AC-110-400 (trade name, manufactured by AsahiChemical Co., Ltd.); and

Electrode liquid: 0.35 mol/L aqueous sodium sulfate solution.

Example 1 Selective Boc-Substitution Reaction in Chitobiitol andChitotriitol and Whole Synthesis of VI-1, VI-2, VII-1 and VII-2)

(1) Synthesis of VI-1

The scheme of synthesis of VI-1 is shown below:

4.16 g (10.0 mmol) of chitobiitol dihydrochloride was measured in a200-mL eggplant flask, dissolved in 80 mL of water and the resultingmixture was stirred at room temperature. 3.40 g (32.0 mmol) of sodiumcarbonate was suspended and 60 mL of methanol was added. 2.29 g (10.5mmol) of dibutyl dicarbonate was added and the resulting mixture wasstirred overnight (for about 16.5 hrs). 1.88 g (11.0 mmol) of Z-chloridewas added to the reaction solution, and agitation was continuedovernight (for about 24 hrs). The solvent of the reaction solution wasevaporated to dryness and colorless residue was obtained. The residualsubstance was dissolved in 60 mL of pyridine, 22 mL of acetic anhydridewas added under stir at room temperature, and the resulting mixture wasstirred at room temperature overnight (for about 23 hrs). 30 mL ofmethanol was added to the reaction solution, and the solvent wasevaporated under reduced pressure. Oily residue was extracted (150 mL ofchloroform×2/200 mL of water), and after the organic layer was driedover anhydrous sodium sulfate, the solvent was evaporated to obtain 9.37g of residual substance. The residual substance was subjected to silicagel chromatography (Silica gel 60 250 g, hexane/ethylacetate/methanol=6/4/1), and 6.06 g (6.95 mmol, yield: 69.5%) of“Compound 4” was obtained.

4.20 g (4.82 mmol) of Compound 4 was measured in a 100 mL eggplantflask, and 421 mg (10 w/w %) of palladium-carbon (act. 10%) and 80 mL ofmethanol were added. The mixture was subjected to catalytic reductionunder hydrogen gas atmosphere under agitation at room temperature. Afterthe reaction ended, filtration with glass filter was carried out, andafter washed with methanol, the solvent of the filtrate was evaporatedand 3.41 g of residual substance was obtained. To this compound, 3.45 g(5.80 mmol) of 3,3-bis(dibenzyloxyphosphoryl)propanoic acid obtained bya known method, for example, the method of Page PCB et al. (Page PCB, etal., J. Org. Chem, 66, 3704-3708, 2001) was added, dissolved in 50 mL ofmethylene chloride, and 929 mg (5.80 mmol) of WSCD and 789 mg (5.84mmol) of HOBtH₂O were added under agitation in an ice bath, andagitation was continued overnight (for about 19.5 hrs). The solvent ofthe reaction liquid was evaporated, the residual substance was subjectedto silica gel chromatography (Silica gel 60 250 g, hexane/ethylacetate/methanol=6/4/1), and 2.41 g (1.83 mmol, yield: 38.0%) of“Compound 6” was obtained.

The following numbers were assigned to carbon atoms in the analysis by¹H-nmr and ¹³C-nmr of “Compound 6.”

¹H-nmr (500 MHz, CDCl₃, δ): 1.4(9H, s, —C(CH₃) 3), 1.82(3H, s, —OAc),1.94(3H, s, —OAc), 1.98(3H, s, —OAc), 1.99(3H, s, —OAc), 2.02(3H, s,—OAc), 2.10(3H, s, —OAc), 2.64˜2.84(1H, 9′), 3.30˜3.44(2H, 8′),3.62˜3.70(1H, 5′), 3.88˜3.96(1H, 1), 3.88˜3.96(1H, 4), 5.22˜5.36(1H, 5),3.88˜3.96(1H, 2′), 4.06˜4.20(1H, 1), 4.06˜4.20(1H, 6), 4.06˜4.20(2H,6′), 4.26˜4.38(1H, 2), 4.46˜4.54(1H, 6), 4.88˜5.20(1H, 3), 4.88˜5.20(1H,4′), 4.88˜5.20 (8H, m, —OCH₂—), 5.10˜5.16(1H, 1′), 5.22˜5.36(1H, 3′),7.3(20H, m, −φ).

¹³C-nmr (500 MHz, CDCl₃, δ): 20.45(—COCH₃), 20.54(—COCH₃),20.65(—COCH₃), 20.70(—COCH₃), 28.27(—COCH₃), 31.68(br s, 8′), 32.13(t,J=Hz, 9′), 48.90, 55.20, 62.32, 62.40, 63.11, 68.10, 68.15, 68.24,68.29, 68.35, 68.40, 68.64, 69.35, 69.43, 69.53, 72.14, 72.28, 74.25,80.30, 98.97(1′), 127.97(−Aromatic), 128.11(−Aromatic), 128.26(−Aromatic), 128.44(−Aromatic), 135.95(d, J=Hz, −Aromatic), 155.77,169.33, 169.59, 169.70, 169.76, 170.27, 170.45, 170.54, 170.71, 170.78.

600 mg (0.46 mmol) of Compound 6 was measured in a 50-mL eggplant flask,dissolved in 20 mL of methanol, and 126 mg (2.33 mmol) of sodiummethoxide was added under agitation at room temperature. After thereaction ended, the reaction liquid was passed through 4.57 g ofAG™-50W-X8 resin (trade name, manufactured by Nippon Bio-RadLaboratories, Inc.), washed out with methanol and water, and the solventwas evaporated to obtain 460 mg (0.451 mmol as Compound 7) of residualsubstance. The residual substance (Compound 7) was dissolved in 7 mL ofmethanol, and 4.0 mL of 10% hydrochloric acid-methanol was added underagitation at room temperature. After 3 hours, the solvent was evaporatedand 392 mg (0.427 mmol as Compound 8) of residual substance wasobtained. To 392 mg (0.427 mmol as Compound 8) of residue, DTPA wasintroduced by a known method, for example, the method of Takahashi etal. [Japanese Patent Laid-Open 2002-187948]. It was dissolved in 7.6 mLof water and 2.41 g of 8 N sodium hydroxide, and warmed to 80° C. Whilekeeping the temperature constant, 1.61 g (4.51 mmol) of anhydrous DTPAwas added to the solution for 3 minutes and warming under agitation wascontinued for 30 minutes. After cooled to 30° C., 0.71 g of 8 N sodiumhydroxide was added to adjust pH to 9.0, and it was warmed to 80° C.again, and warming under agitation was carried out for 30 minutes. Then,the mixture was cooled to 30° C., and 0.5 mL of 6 N hydrochloric acidwas added to adjust pH to 8.0 to terminate the reaction. The solvent ofthe reaction liquid was evaporated, 9.5 mL of water was added todissolve it and 831 mg (200 w/w %) of palladium-carbon (act. 10%) wasadded and the mixture was subjected to catalytic reduction underhydrogen gas atmosphere for 1.5 hours under agitation at roomtemperature. After the reaction ended, filtration with glass filter wascarried out, the solvent of the filtrate was evaporated, and the residuewas obtained. The residue was separated and purified by recycling HPLC,and after desalination treatment, the solvent was evaporated and 115 mg(0.123 mmol; yield: 27.0%) of “Compound 10; VI-1” was obtained.

The following numbers were assigned to carbon atoms in the analysis by¹H-nmr and ¹³C-nmr of “Compound VI-1.”

¹H-nmr(500 MHz, D₂O, TSP): 2.30˜2.50(9′), 2.60˜2.80(8′), 3.1˜3.4(DTPA),3.39˜3.46 (5′), 3.46˜3.52(4′), 3.52˜3.60(5), 3.60˜3.69(1),3.61˜3.69(3′), 3.70˜3.75(2′), 3.70˜3.75(6), 3.70˜3.83(1), 3.75˜3.82(4),3.75˜3.82(6′), 3.83˜3.87(6), 3.87˜3.92(3), 3.87˜3.93(6′), 4.35˜4.40(2),4.62˜4.68(1′), 8.4(7 or 7′), 8.7(7′ or 7).

¹³C-nmr (500 MHz, D₂O, TSP): 33.49(8′), 36.41(t, J=136 Hz; 9′), 51.16,51.74, 52.00, 53.12, 55.85, 56.67, 57.90, 58.16, 58.43, 60.74, 61.22,62.29, 68.13, 69.30, 71.30, 73.85, 75.86, 79.01, 101.14(1′), 172.02,173.92, 174.31, 175.93, 175.98, 176.02, 176.07, 176.85.

MS(ESI−): 932.2427 C₂₉H₅₂N₅O₂₅P₂ requires 932.2477([M−H]⁻).

(2) Synthesis of VI-2

The scheme of synthesis of VI-2 is shown below:

15.0 g (36.1 mmol) of chitobiitol dihydrochloride was measured anddissolved in 280 mL of water, and 12.3 g (116 mmol) of sodium carbonatewas suspended, and 210 mL of methanol was added, and the mixture wasstirred at room temperature. 8.71 mL (38.0 mmol) of dibutyl dicarbonatewas added and the resulting mixture was stirred overnight at roomtemperature. 5.7 mL (40.0 mmol) of Z-chloride was added, and agitationwas conducted overnight at room temperature. The solvent of the reactionliquid was evaporated, 217 mL of pyridine was added to the residualsubstance, and 79.4 mL (842 mmol) of acetic anhydride and a catalyticamount of 4-(N,N-dimethylamino) pyridine were added in an ice bath, andthe resulting mixture was stirred overnight. 108 mL of methanol wasadded to the reaction solution in an ice bath, and the solvent wasevaporated. The residue was extracted (chloroform/saturated potassiumbisulfate aqueous solution, saturated sodium bicarbonate aqueoussolution), and the organic layer was washed with water and dried overanhydrous sodium sulfate, and filtrated after the solvent was thenevaporated. The obtained crude product was subjected to columnchromatography (Silica gel N60 (trade name, manufactured by KantoChemical Co., Inc.), ethyl acetate/hexane/methanol=4/6/1), and thesolvent was evaporated to obtain 18.3 g (21.0 mmol, yield: 58.3%) of“Compound 4.”

4.35 g (5.0 mmol) of Compound 4 was measured, and dissolved in 50 mL ofmethanol, and 1.35 g (25.0 mmol) of sodium methoxide was added, andagitation at room temperature was carried out for 2 hours. 50.0 g ofwashed AG™-50W-X8 resin (trade name, manufactured by Nippon Bio-RadLaboratories, Inc.) was added. It was succeedingly stirred for 30minutes and filtered, and the solvent of the filtrate was evaporated. Itwas redissolved in 21 mL of methanol, and 12 mL of 10% hydrochloricacid-methanol solution (manufactured by Tokyo Kasei Kogyo Co., Ltd.) wasadded. The solvent was evaporated and the residual substance wasobtained after carrying out agitation at room temperature for 3 hours.3.57 g (6.00 mmol) of 3,3-bis(dibenzyloxyphosphoryl)propanoic acid wasadded to this residual substance, and 50 mL of dimethylformamide wasadded and was stirred. 1.10 mL (6.26 mmol) of WSCD and 811 mg (6.00mmol) of HOBt-H₂O were added one by one under argon atmosphere in an icebath, and the mixture was stirred overnight. The solvent was evaporated,30 mL of pyridine was added, a catalytic amount of4-(N,N-dimethylamino)pyridine was added, and 12.0 mL (127 mmol) ofacetic anhydride was added. After it was stirred overnight, 15 mL ofmethanol was added and extraction (chloroform/saturated potassiumbisulfate aqueous solution, saturated sodium bicarbonate aqueoussolution) was performed. After the organic layer was washed with waterand dried over anhydrous sodium sulfate, the solvent was evaporated toobtain the residual substance (about 7.7 g). This residual substance wassubjected to column chromatography (Silica gel N60 (trade name,manufactured by Kanto Chemical Co., Inc.) about 200 g, hexane/ethylacetate/methanol=4/6/1), and the crude product fractions 1, 2, and 3were obtained. Fractions 1 and 2 were subjected to preparative TLC (Cat.No. 105717 manufactured by Merck Co., hexane/ethylacetate/methanol=5/5/1), and 741.6 mg (490.9 mg from Fraction 1, 250.7mg from Fraction 2) of “Compound 8” was obtained. Fraction 3 was againsubjected to column chromatography (Silica gel N60 (manufactured byKanto Chemical Co., Inc.) about 100 g, hexane/ethylacetate/methanol=10/5/1), and 1.17 g (0.498 mmol, yield: 14.6%) of“Compound 8” was obtained. 1.91 g (1.42 mmol, yield: 28.4%) of “Compound8” was obtained from this reaction in total.

1.10 g (0.816 mmol) of Compound 8 was dissolved in 10 mL of methanol,and 309 mg (5.71 mmol) of sodium methoxide was added under argonatmosphere, and was stirred at room temperature for 2 hours. 9.0 g ofAG™-50W-X8 resin (trade name, manufactured by Nippon Bio-RadLaboratories, Inc.) was added and agitation was continued for 30minutes. The reaction mixture was filtered, the solvent of the filtratewas evaporated and 551 mg of residual substance was obtained. 15 mL ofmethanol and 5 mL of saturated sodium bicarbonate aqueous solution wereadded to the residual substance. 400 mg of palladium-carbon (act. 10%)was added, and the mixture was stirred under hydrogen atmosphere, andsubjected to catalytic reduction for 2 hours. After the reaction ended,it was filtered and the filtrate was evaporated. 5 mL of water and 4.17mL of 8 mol/L sodium hydroxide aqueous solution were added to thisresidual substance and the mixture was stirred. It was heated to 80° C.,2.97 g (8.20 mmol) of anhydrous DTPA was added, and agitation wascontinued for 30 minutes. After allowed to room temperature, 8 mol/Lsodium hydroxide aqueous solution was added to adjust to pH 9, and themixture was warmed to 80° C. again for 30 minutes. It was allowed toroom temperature, and adjusted to pH 8 and concentrated. Thisconcentrated liquid was separated and purified by recycling HPLC(column: Shodex Asahipak GS 320-21G (trade name, manufactured by ShowaDenko K.K., 21.5 mm ID×500 mm) and Shodex Asahipak GS 220-21G (tradename, manufactured by Showa Denko K.K., 21.5 mm ID×500 mm) seriallyconnected, mobile phase: 100 mmol/L sodium chloride aqueous solution,flow rate: 5.0 mL/min, detector: visible-ultraviolet absorptiometer(detection wavelength: 210 nm)). Desalting was performed on theseparated solution, and the solvent was evaporated to isolate 239.9 mg(0.257 mmol, yield: 31.5%) of “Compound VI-2.”

The following numbers were assigned to carbon atoms in the analysis by¹H-nmr and ¹³C-nmr of “Compound VI-2.”

¹H-nmr (500 Hz, D₂0, TSP): 3.80(1H, m, 1), 3.65(1H, m, 1), 4.30(1H, m,2), 3.89(1H, m, 3), 3.82(1H, m, 4), 3.83(1H, m, 5), 3.71(1H, m, 6),3.53(1H, m, 6), 4.66(1H, d, J=8.2 Hz, 1′), 3.79(1H, m, 2′), 3.71(1H, m,3′), 3.51(1H, m, 4′), 3.48(1H, m, 5′), 3.92(1H, m, 6′), 3.81(1H, m, 6′),2.72(dd, ³J_(H-P)=15.1 Hz, J=6.9 Hz, —COCH ₂CH—), 2.39 (dd,2J_(H-P)=21.1 Hz, J=6.9 Hz, —COCH₂CH—), 3.65(2H, m), 3.62(2H, s),3.39(2H, s), 3.62(2H, s), 3.29(2H, s), 3.22(2H, m), 3.08(2H, m).

¹³C-nmr (500 MHz, D₂0, TSP): 61.30(1), 53.67(2), 68.57(3), 78.47(4),71.42(5), 62.33(6), 101.28(1′), 56.00(2′), 73.56(3′), 69.98(4′),75.86(5′), 60.77(6′), 176.01(t, ³J_(C-P)=8.6 Hz), 33.70, 36.78(t,²J_(C-P)=155.2 Hz), 173.66, 177.86, 174.12, 174.66, 58.65, 58.73, 56.00,58.10, 52.35, 51.51, 51.12.

³¹P-nmr (500 MHz, D₂0): 19.43, 19.38.

MS (ESI−): 1066.1454 C₂₉H₄₈N₅O₂₅P₂Na₆ requires 1466.1500 [[M·Na₅+Na]⁻.

(3) Synthesis of VII-1

The scheme of synthesis of VII-1 is shown below:

3.08 g (5.02 mmol) of chitotriitol trichloride was measured in a 200 mLeggplant flask, 2.24 g (21.10 mmol) of sodium carbonate was added, and50 mL of methanol and 36 mL of water were added and the mixture wasstirred. 1.4 mL (6.09 mmol) of dibutyl dicarbonate was added, then,after stirring for 14 hours the solvent of the reaction mixture solutionwas evaporated. 58 mL of methanol was added to the residual substanceand was stirred, and 1.45 mL (11.92 mmol) of p-methoxybenzaldehyde wasadded, and the mixture was stirred for 24 hours. The solvent of thereaction mixture solution was evaporated, 43 mL of pyridine was added tothe residual substance, and it was stirred in a water bath, and 11.3 mL(11.98 mmol) of acetic anhydride was added, and it was stirred for 17hours. 21 mL of methanol was added on an ice bath and agitation wascontinued for 15 more minutes. The solvent of the reaction mixturesolution was evaporated, and extracted (150 mL of chloroform/100 mL ofwater×2) organic layer was dried over anhydrous sodium sulfate, and thesolvent was evaporated. 20 mL of dimethylformamide was added to theresidue, and after the residue was dissolved, the solvent was evaporatedand the residual substance (about 7.2 g) was obtained. 25 mL of ethylacetate was added to the residual substance, 25 mL of 1 mol/Lhydrochloric acid was added under agitation at room temperature, andagitation was continued for 1 hour. The reaction liquid was washed withethyl acetate (25 mL×4), a basic aqueous solution (15 mL of 5% sodiumacetate aqueous solution+35 mL of saturated sodium carbonate aqueoussolution) was added, and adjusted to pH 8 and extracted (chloroform 50mL×2). The organic layer was washed with 100 mL of saturated sodiumbicarbonate, dried over anhydrous sodium sulfate, and the solvent wasevaporated to obtain 3.09 g (3.14 mmol, yield: 62.7%, pale yellowcrystal) of “Compound 5.”

5.62 g (9.45 mmol) of 3,3-bis(dibenzylphosphoryl)propanoic acid and 853mg (6.31 mmol) of HOBt.H₂O were added to 3.09 g (3.14 mmol) of “Compound5,” and dissolved in 70 mL of dimethylformamide. 1.65 mL (9.39 mmol) ofWSCD was added in an ice bath under agitation, and the resulting mixturewas stirred for 17 hours. The solvent of the reaction mixture solutionwas evaporated and extracted (chloroform 150 mL/saturated sodiumbicarbonate aqueous solution 100 mL, water 100 mL+saturated sodiumchloride aqueous solution 20 mL×3, saturated sodium chloride aqueoussolution 100 mL), and the organic layer was dried over anhydrous sodiumsulfate and the solvent was evaporated and the residual substance (about7.4 g, pale yellow crystal) was obtained. The residual substance wassubjected to column chromatography (Silica gel N60 (trade name,manufactured by Kanto Chemical Co., Inc.) 250 g,chloroform/methanol=20/1), and the crude product (Fr 2-3; 1.30 (yellowoily substance) and Fr 4-6; 842 mg (pale yellow crystal)) were obtainedfrom Fractions 2 to 3 (about 600 to 900 mL; 300 mL) and Fractions 4 to 6(about 900 to 1200 mL; 300 mL). After the crude product of Fraction 2 to3 was subjected to column chromatography (Silica gel N60 (trade name,manufactured by Kanto Chemical Co., Inc.) 100 g, hexane/ethylacetate/methanol=12/8/1) to elute high polarity impurities by 1000 mL ofmobile phase and then the object fraction was eluted bychloroform/methanol=10/1, and 831 mg (pale yellow crystal) of “Compound6” was obtained. In the meantime, the crude product of Fractions 4 to 6was subjected to column chromatography (Silica gel N60 (trade name,Manufactured by Kanto Chemical Co., Inc.) 100 g, ethylacetate/chloroform/methanol=10/5/1) and 286 mg (pale yellow crystal) of“Compound 6” was obtained from fractions 11 to 13 (ca. 280 to 340 mL).1.12 g (0.523 mmol, yield: 16.6%) of “Compound 6” was obtained from thisreaction in total.

The following numbers were assigned to carbon atoms in the analysis by¹H-nmr of “Compound 6.”

¹H-nmr(500 MHz, CDCl₃, δ): 4.42(1H, m, 1), 3.92(1H, m, 1), 4.32(1H, m,2), 5.05(1H, m, 3), 3.94(1H, m, 4), 5.29(1H, m, 5), 4.49(1H, dd, J=12, 2Hz, 6), 4.12 (1H, dd, J=12, 6 Hz, 6), 4.92 (1H, d, J=10 Hz, 2-NH),4.81(1H, br.d, H=7 Hz, 1′), 3.94(1H, m, 2′), 5.09(1H, t, J=10 Hz, 3′),3.63(1H, t, J=10 Hz, 4′), 3.46(1H, m, 5′), 4.31(1H, m, 6′), 4.25(1H, m,6′), 4.53(1H, d, J=8 Hz, 1″), 3.88(1H, m, 2″), 5.16(1H, t, J=10 Hz, 3″),5.02 (1H, m, 4″), 3.60(1H, m, 5″), 4.36(1H, dd, J=13, 4 Hz, 6″),3.99(1H, dd, J=13, 2 Hz, 6″), 6.57(1H, d, J=9 Hz, 2″-NH), 2.08(3H, s,6′-Ac), 2.05(3H, s, 6″-Ac), 2.03(3H, s, 1-Ac), 1.982(3H, s, 4″-Ac),1.977(3H, s, 6-Ac), 1.96(3H, s, 3-Ac), 1.95(3H, s, 5′-Ac), 1.85(3H, s,3′-Ac), 1.81(3H, s, 3″-Ac), 1.41 (9H, s, C(CH₃)₃), 2.68˜2.89((2H, m,—COCH₂CH—), 3.28˜3.46(1H, m, —COCH₂CH—), 7.24˜7.32(20H, —CH₂—Ar),4.92˜5.05 (8H, —CH₂—Ar)

¹³C-nmr (500 MHz, CDCl₃, δ): 63.18, 49.02, 69.44, 74.01, 69.29, 62.30,99.14, 54.86, 72.52, 75.95, 72.80, 62.10, 100.95, 54.86, 72.25, 67.89,71.79, 61.51, 171.31(6′-Ac), 170.90(3″-Ac), 170.66(3′-Ac), 170.49(1-Ac,6″-Ac), 170.46(3-Ac, 5-Ac), 169.53(6-Ac), 169.24(4″-Ac),20.38˜20.68(—CH₃), 155.51, 80.01, 28.23, 169.44˜169.64, 31.69(m),31.49(m), 32.08(t, J_(CP)=135 Hz), 31.98(t, J_(CP)=134 Hz),135.80˜136.05(—CH₂—Ar), 127.80˜128.05(—CH₂—Ar), 68.05˜68.35(—CH₂—Ar)

³¹P-nmr (500 MHz, CDCl₃): 24.54(d, J_(PP)=3 Hz), 24.19(d, J_(Pp)=4 Hz),24.15(d, J_(PP)=3 Hz), 24.11(d, J_(PP)=4 Hz).

650 mg (3.28 mmol) of Compound 6 was dissolved in 20 mL of methanol in a100 mL eggplant flask, and 138 mg (2.65 mmol) of sodium methoxide wasadded under argon atmosphere, and the mixture was stirred at roomtemperature for 1 hour. 4.0 g of AG™-50W-X8 resin (trade name,manufactured by Nippon Bio-Rad Laboratories, Inc.) was added and 20 mLof water was added, and agitation was continued for 15 minutes. Thereaction liquid was filtered, the filtrate was evaporated and 551 mg ofresidual substance was obtained. The residual substance was dissolved in10 mL of methanol, 5 mL of 10% hydrochloric acid-methanol (manufacturedby Tokyo Kasei Kogyo Co., Ltd.) was added, and agitation at roomtemperature was carried out for 3 hours. The solvent of the reactionliquid was evaporated and 481 mg of residual substance was obtained.4.43 mL of water and 1.63 mL of 8 mol/L sodium hydroxide aqueoussolution were added to this residual substance and the mixture wasstirred. It was heated to 80° C., 1.11 g (3.11 mmol) of anhydrous DTPAwas added, and agitation was continued for 30 minutes. It was allowed toroom temperature, and 8 mol/L of sodium hydroxide aqueous solution wasadded, adjusted to pH 9, and warmed to 80° C. again for 30 minutes. Itwas allowed to room temperature and adjusted to pH 8. The reactionliquid was washed with chloroform to remove insoluble matters, and acatalytic amount of palladium-carbon (act. 10%) was added to the aqueousphase, and the resulting mixture was stirred under hydrogen atmosphereand subjected to catalytic reduction for 3 hours. After the reactionended, filtration was performed and the filtrate was condensed. Thiscondensed liquid was separated and purified by recycling HPLC (column:Shodex Asahipak GS 320-21G (trade name, manufactured by Showa DenkoK.K., 21.5 mm ID×500 mm) and Shodex Asahipak GS 220-21G (trade name,manufactured by Showa Denko K.K., 21.5 mm ID×500 mm) serially connected,mobile phase: 100 mmol/L sodium chloride aqueous solution, flow rate:5.0 mL/min, detector: visible-ultraviolet absorptiometer (detectionwavelength: 210 nm)). Desalting was performed on the separated solution,and the solvent was evaporated to isolate 119 mg (0.09 mmol, yield:29.7%) of “Compound VII-1.”

The following numbers were assigned to carbon atoms in the analysis by¹H-nmr of “Compound VII-1.”

¹H-nmr (500 Hz, D₂0, TSP): 3.81(1H, m, 1), 3.68(1H, m, 1), 4.38(1H, m,2), 3.91(1H, m, 3), 3.70(1H, m, 4), 3.88(1H, m, 5), 3.79(1H, m, 6),3.57(1H, m, 6), 4.64(1H, d, J=8 Hz, 1′), 3.82(1H, m, 2′), 3.76(1H, m,3′), 3.71(1H, m, 4′), 3.54(1H, m, 5′), 3.91(1H, m, 6′), 3.72(1H, m, 6′),4.69(1H, d, J=8 Hz, 1″), 3.73(1H, m, 2″), 3.67(1H, m, 3″), 3.47(1H, m,4″), 3.51(1H, m, 5″), 3.92(1H, m, 6″), 3.75(1H, m, 6″), 4.00(2H, s,—NHCOCH₂—), 3.75(2H, s, —NCH₂CO₂H), 3.62(2H, s, —NCH₂CO₂H), 3.89(4H, s,—NCH₂CO₂H×2), 3.39(2H, m, —NCH₂CH₂N—), 3.23(2H, m, —NCH₂CH₂N—), 3.32(2H,m, —NCH₂CH₂N—), 3.56(2H, m, —NCH₂CH₂N—), 2.77(2H, m, —COCH₂CH—),2.57(1H, m, —CH₂CH—).

¹³C-nmr(500 Hz, D₂0, TSP): 61.09, 53.38, 68.22, 78.82, 71.30, 62.29,101.07, 56.04, 72.53, 78.33, 74.66, 60.29, 100.95, 56.65, 73.91, 69.58,76.20, 60.84, 168.60, 172.80, 174.30, 170.65, 57.49, 55.26, 57.63,52.53, 51.06, 50.00, 52.25, 175.19˜175.31(m), 33.28, 33.12, 36.18(t,J_(CP)=189 Hz), 35.67 (t, J_(CP)=120 Hz).

³¹P-nmr (500 Hz, D₂0): 19.60, 19.39, 19.31, 18.85.

(4) Synthesis of VII-2

The scheme of synthesis of VII-2 is shown below:

10.0 g (16.3 mmol) of chitotriitol trichloride salt was measured in a300 mL eggplant flask, dissolved in 160 mL of water and stir at roomtemperature was carried out. 16.4 g (155 mmol) of sodium carbonate wassuspended, and 80 mL of methanol was added. 5.34 g (24.5 mmol) ofdibutyl dicarbonate was added, and the mixture was stirred at roomtemperature for 41 hours. 13.1 g (81.6 mmol) of Z-chloride was added,and the mixture was stirred at room temperature further for about 24.5hours. After the solvent of the reaction liquid was evaporated, theresidual substance was suspended in 100 mL of pyridine. 50 mg of4-(N,N-dimethylamino)pyridine which is a catalytic amount and 40.0 g(392 mmol) of acetic anhydride were added to this in an ice bath, thereaction liquid was allowed to room temperature, and the mixture wasstirred for 21 hours. After 10 mL of methanol was added to the reactionsolution in an ice bath and agitated for 10 more minutes, the mixturewas poured into 300 mL of saturated sodium bicarbonate aqueous solutionby small portions, and extracted (chloroform 200 mLx3). After theorganic layer was washed with 50 mL of a saturated sodium chloridesolution and dried over anhydrous sodium sulfate, it was filtered andthe solvent was evaporated. 23.4 g of the obtained crude product wassubjected to column chromatography (Silica gel N60 (trade name,manufactured by Kanto Chemical Co., Inc.) 250 g, ethylacetate/hexane/methanol=8/12/1→8/12/2), and 15.2 g (12.2 mmol, yield:74.5%) of “Compound 4” was obtained.

4.26 g (3.41 mmol) of Compound 4 was measured in a 100 mL eggplantflask, and dissolved in 50 mL of methanol, 56.6 mg (1.05 mmol) of sodiummethoxide was added, and agitation at room temperature was carried outfor 2 hours. After 5.0 g of washed AG™-50W-X8 resin (trade name,manufactured by Nippon Bio-Rad Laboratories, Inc.) was added and theresulting mixture was stirred for 20 minutes succeedingly, filterfiltration was carried out, and the solvent of the filtrate wasevaporated. 80 mL of methanol was redissolved under argon atmosphere,and 20.0 mL of 10% hydrochloric acid-methanol (manufactured by TokyoKasei Kogyo Co., Ltd.) was added, and after agitation was carried out atroom temperature for 1 hour, the solvent was evaporated and 3.09 g ofresidual substance was obtained. 2.56 g (4.31 mmol) of3,3-bis(dibenzyloxyphosphoryl)propanoic acid was measured in a 100 mLeggplant flask, and 3.09 g of the above residual substance dissolved in30 mL of dimethylformamide was added thereto and the mixture wasstirred. 0.75 mL (5.47 mmol) of WSCD and 555 mg (4.10 mmol) of HOBt-H₂Owere added one by one at room temperature, and agitation at roomtemperature was carried out under argon atmosphere for 69 hours. Thesolvent was evaporated, 30 mL of pyridine was added and 8.0 mL (84.8mmol) of acetic anhydride and a catalytic amount of4-(N,N-dimethylamino)pyridine were added in an ice bath under agitation.

After 140 hours, 10 mL of methanol was added, and chloroform extractionwas carried out after 15 minutes. After the organic layer was dried overanhydrous sodium sulfate, the solvent was evaporated and the residualsubstance (about 6.9 g) was obtained. This residual substance wassubjected to column chromatography (200 g of Silica gel N60 (trade name,manufactured by Kanto Chemical Co., Inc.), hexane/ethylacetate/methanol=12/8/1→6/4/1→5/5/1), and 859 mg (0.498 mmol, yield:14.6%) of “Compound 8” was obtained.

859 mg (0.498 mmol) of Compound 8 was dissolved in 20 mL of methanol,and 107 mg of palladium-carbon (act. 10%) was added under argonatmosphere. It was stirred under hydrogen atmosphere and subjected tocatalytic reduction for about 4 hours. After the reaction ended, themixture was filtered and the solvent of the filtrate was evaporated. 790mg of sodium carbonate was added to the residue, and 50 mL of water wasadded to dissolve the residue. This solution was stirred at roomtemperature for 22.5 hours, and after adjusted to pH 7 with 1 mol/L ofhydrochloric acid, the solvent was evaporated and the residual substancewas obtained.

To the residual substance, 5.66 mL of 8 mol/L sodium hydroxide and 5.00mL of water were added and the mixture was stirred under nitrogenatmosphere, and warmed to 80° C. 3.75 g (10.5 mmol) of anhydrous DTPAwas added to this, and the mixture was allowed to react for 30 minutes.Then, the reaction solution was allowed to room temperature, and 8 mol/Lsodium hydroxide solution was added to adjust to pH 9, and the mixturewas warmed to 80° C. again and stirred for 30 minutes. Then, thereaction solution was allowed to room temperature, and concentratedhydrochloric acid was dropped to adjust pH to 1.8, and the mixture wasallowed to stand still overnight. The deposited substances were removedby filtration, sodium hydroxide was added to the obtained filtrate, toadjust to pH 8.2, and separation and purification by HPLC was performedon the following conditions.

Column: POROS 50HQ (product name, manufactured by PE biotechnologymedical systems, 30 mmID×240 mm);

Flow rate: 70 mL/min;

Detection: visible-ultraviolet absorptiometer (absorbance: 210 nm);

Elution condition: 150→250 mmol/L NaCl/60 min (gradient elution);

Desalination operation was performed on the separated liquid, thesolvent was evaporated, and 161 mg (0.11 mmol, yield: 22.0%) of“Compound VII-2” was isolated.

¹H-nmr (500 MHz, D₂0, TSP): 2.62(1H, m, —CH₂CH—), 2.83(2H, m,—COCH₂CH—), 3.52-3.97 (54H, m), 4.33(1H, m), 4.72(2H, m).

¹³C-nmr (500 MHz, D₂0, TSP): 33.39, 36.28(t), 50.59 (m), 50.83 (m),52.03((m), 25.16((m), 52.59((m), 52.79((m), 53.40, 55.67, 56.05, 56.68,57.72, 57.97, 58.08, 58.38, 60.33, 60.92, 61.30, 62.21, 68.58, 70.15,71.39, 72.34, 73.57, 74.69, 76.16, 78.55, 79.15, 100.90, 170.90, 171.06,171.32, 171.48, 174.78, 174.95, 175.09, 175.15, 175.22.

³¹P-nmr(500 MHz, D₂O): 19.13, 19.21.

MS (ESI−): 1467.4328 C₄₉H₈₃N₉O₃₈P₂ requires 1467.4314.

Example 2 Labeling of VI-1

(1) 111-Indium Labeling

A kit was prepared by adjusting the synthesized-compound VI-1 to 1.5μmol/0.5 mL by adding thereto sodium acetate buffer solution (pH 4.4),and 0.5 mL of indium chloride (¹¹¹InCl₃) was added, and the mixture wasallowed to stand at room temperature to perform the labeling reaction.The mixture was analyzed by silica gel TLC using 10% ammoniumacetate/methanol as a developing solvent. Consequently, ¹¹¹InCl₃ was notobserved and it was confirmed that VI-1 had been labeled with ¹¹¹In.

(2) 99m-Technetium Labeling

The synthesized-compound VI-1 was mixed with a stannous chloride aqueoussolution. A physiological saline containing pertechnetate of 17 to 20mCi was added to the mixture, and the mixture was allowed to stand stillat room temperature to perform the labeling reaction.

Example 3 Study of In Vivo Distribution

The sample was administered via the tail vein under ravonal anesthesiato SD rats (female, 8 to 9-week age, n≧3) under non-fasting. At eachtime point after administration, blood was collected from the abdominalaorta to allow the animal to die due to the loss of blood, the organswere isolated, radioactivity was counted and organ weights weremeasured, and the distribution was computed. The measurement results areshown as % ID/g for organ and as % ID for urine. TABLE 1 In vivodistribution of 99 mTc-labeled VI-1 Organs 1-hour point 2-hour pointBlood 0.038 ± 0.009 0.014 ± 0.002 Lower limb bone 2.826 ± 0.167 2.956 ±0.114 Liver 0.024 ± 0.005 0.018 ± 0.005 Kidney 1.281 ± 0.997 0.992 ±0.797 Urine (% ID) 57.598 ± 7.869  62.899 ± 2.590 

TABLE 2 In vivo distribution of 99 mTc-MDP injection (comparative andreferential example) Organs 1-hour point Blood 0.068 ± 0.012 Lower limbbone 2.649 ± 1.835 Liver 1.389 ± 0.296 Kidney 2.649 ± 1.835 Urine (% ID)44.674 ± 2.565 

As shown above, excretion into urine was rapid and blood clearance wasalso rapid. As for affinity with calcified tissues, high accumulationwas observed. In addition, in consideration of the fact that thecomposition of the present invention in the above-mentioned example wasnot purified nor optimized by any means in remarkable contrast to thecommercially available composition MDP for injection shown above as acontrol, it will be apparent that the optimized composition of thepresent invention will exhibit still more dramatic superiority.

Example 4 Selective Boc-Substitution Reaction in Chitotriitol

Selective Boc-substitution reaction in chitotriitol was carried andderived as shown in the following scheme.

Chitotriitol trichloride (3.06 g, 5.00 mmol) was dissolved in 20 mL ofmethanol and 40 mL of water, and 2.23 g (21.1 mmol) of sodium carbonateand 1.31 g (6.00 mmol) of dibutyl dicarbonate were added. This reactionmixture was stirred at room temperature for 22 hours, and the solventwas condensed under reduced pressure. The obtained residual substancewas dissolved in 50 mL of methanol, 1.63 g (12.0 mmol) of p-anisaldehydewas added, and the mixture was stirred at room temperature for 24 hours.Subsequently, the reaction liquid was concentrated under reducedpressure and after the obtained residual substance was washed withhexane, it was dried under reduced pressure at room temperatureovernight. This residual substance was suspended in 30 mL of pyridine,and 12.3 g (120 mmol) of acetic anhydride was dropwised in an ice bath.The reaction liquid was allowed to room temperature, and after 17.5 houragitation, 10 mL of methanol was added and agitation was performed for40 more minutes in an ice bath, and then it was poured into an icedwater (about 800 mL) under agitation. The deposited precipitate wasfiltered, dried under reduced pressure at room temperature overnight,and 5.34 g (yield: 87.7%, white powder) of the target compound wasobtained.

¹H-nmr (270 MHz, CDCl₃, δ): 1.40 (s, 9H), 1.85 (s, 3H), 1.91 (s, 3H),1.92 (s, 3H), 1.99 (s, 3H), 2.01 (s, 3H), 2.03 (s, 3H), 2.05 (s, 3H),2.10 (s, 3H), 2.11 (s, 3H), 3.28 (q, J=8.6 Hz, 2H), 3.51 (br d, 1H),3.84 (s, 3H), 3.85 (s, 3H), 3.88-3.78 (m, 1H), 4.12-3.96 (m, 4H),4.60-4.21 (m, 6H), 4.82-4.71 (m, 3H), 4.98 (d, J=9.2 Hz, 1H), 5.06 (t,J=9.6 Hz, 2H), 5.20 (t, J=9.6 Hz, 1H), 5.30 (t, J=9.6 Hz, 1H), 6.91 (d,J=8.6 Hz, 2H), 6.93 (d, J=8.6 Hz, 2H), 7.64 (d, J=8.6 Hz, 2H), 7.67 (d,J=8.6 Hz, 2H), 8.07 (s, 1H), 8.09 (s, 1H).

INDUSTRIAL APPLICABILITY

According to the present invention, a compound in which a group AChaving affinity with a calcified tissue is bonded to a mother core MChaving a controlled molecular size is provided. This compound showsexcellent affinity with a calcified tissue, and the compound which failsto accumulate in the calcified tissues exhibit high excretability inurine. Furthermore, a ligand LI can be bonded to the mother nucleus MC,so that the function of labeling by complex formation or the like isassigned to the ligand LI, the group AC having affinity with calcified atissue is prevented from forming a complex, and thereby clearance fromblood and/or soft tissues and excretion into urine are further promoted.

1. A compound having affinity with a calcified tissue represented by the formula: (AC)_(a)-MC-(LI)_(b) wherein MC is a mother nucleus and represents a residue of a compound having a plurality of functional groups selected from the group consisting of an amino group, an amide group, a hydroxyl group, a thiol group, a thioether group, a sulfonyl group, a phosphonyl group, an aldehyde group, a carboxyl group, a carbonyl group, a halogen, and a cyano group; AC is a group having affinity with a calcified tissue; LI is a ligand for binding to a metal atom; and a is an integer of I or more, and b is 0 or an integer of 1 or more.
 2. The compound having affinity with a calcified tissue according to claim 1, wherein the mother nucleus MC is a residue of a compound selected from the group consisting of a monosaccharide, an oligosaccharide, an amino oligosaccharide, a cyclodextrin and a saccharide dendrimer.
 3. The compound having affinity with a calcified tissue according to claim 1, wherein the AC is selected from the group consisting of polyaspartic acid, polyglutamic acid and organic phosphonic acid.
 4. The compound having affinity with a calcified tissue according to claim 1, wherein the mother nucleus MC is a residue of a compound selected from the group consisting of an oligosaccharide, an amino oligosaccharide, a cyclodextrin and a saccharide dendrimer, and the group AC having affinity with a calcified tissue is bonded to a constituent monosaccharide of the mother nucleus MC, and the ligand LI for binding to a metal atom is bonded to a constituent monosaccharide other than the above-mentioned constituent monosaccharide.
 5. The compound having affinity with a calcified tissue according to claim 4, wherein a plurality of the groups AC having affinity with a calcified tissue or a plurality of the ligands LI for binding to a metal atom are bonded to the mother nucleus MC.
 6. The compound having affinity with a calcified tissue according to claim 1, wherein at least one of the mother nucleus MC, the group AC having affinity with a calcified tissue and the ligand LI contains a metal atom or an isotope of a halogen atom, carbon, oxygen, nitrogen, sulfur or phosphorus.
 7. The compound having affinity with a calcified tissue according to claim 1, which forms a complex with a metal atom.
 8. The compound having affinity with a calcified tissue according to claim 1, wherein the mother nucleus MC is a residue of a linear or branched oligosaccharide of 2 to 20 saccharide units which comprises a constituent monosaccharide selected from the group consisting of glucose, mannose and galactose.
 9. The compound having affinity with a calcified tissue according to claim 1, wherein the mother nucleus MC is a residue of a linear or branched amino oligosaccharide of 2 to 20 saccharide units which comprises a constituent monosaccharide selected from the group consisting of glucosamine, mannosamine and galactosamine.
 10. The compound having affinity with a calcified tissue according to claim 9, wherein a part of the amino oligosaccharide that constitutes the mother nucleus MC is reduced.
 11. The compound having affinity with a calcified tissue according to claim 9, wherein a part of the amino oligosaccharide that constitutes the mother nucleus MC is N-acetylated.
 12. The compound having affinity with a calcified tissue according to claim 8, wherein the oligosaccharide or amino oligosaccharide comprises constituent monosaccharides that are α- or β-linked.
 13. The compound having affinity with a calcified tissue according to claim 8, wherein the oligosaccharide or amino oligosaccharide comprises constituent monosaccharides that are 1-3, 14 or 1-6-linked.
 14. The compound having affinity with a calcified tissue according to claim 1, wherein the mother nucleus MC comprises a residue of a cyclodextrin selected from the group consisting of α-, β- and γ-cyclodextrins.
 15. The compound having affinity with a calcified tissue according to claim 14, wherein the cyclodextrin is a dialdehyde saccharide which comprises a constituent monosaccharide that is reduced at positions 2 and
 3. 16. The compound having affinity with a calcified tissue according to claim 1, wherein the mother nucleus MC comprises a residue of a saccharide dendrimer, and the saccharide dendrimer comprises a linear or branched saccharide bonded to a core comprising a polycarboxylic acid or an alkyl polycarboxylic acid.
 17. The compound having affinity with a calcified tissue according to claim 1, wherein the mother nucleus MC comprises a residue of a saccharide dendrimer, and the saccharide dendrimer comprises a linear or branched saccharide bonded to a core comprising a polyamine or an alkylpolyamine.
 18. The compound having affinity with a calcified tissue according to claim 1, wherein the group AC having affinity with a calcified tissue comprises an organic phosphonic acid, and the organic phosphonic acid is a residue of a diphosphonic acid represented by the following formula I, derivatives thereof or salts thereof:

(wherein, R¹ and R³, which are the same or different, each represents a formula —(CR⁵R⁶)_(k)—R⁷ _(l)—(CR⁸R⁹)_(m)—R¹⁰ _(n)—(CR¹¹R¹²)_(o)—R¹³ _(p)—(CR¹⁴R¹⁵)_(q)R¹⁶ (wherein R⁵, R⁶, R⁸, R⁹, R¹¹, R¹², R¹⁴, R¹⁵ and R¹⁶ are groups each independently selected from the group consisting of H, —OH, —COOH, —C(NH₂)═NH, —CN, —SO₃H, —NR¹⁷ ₂ and a halogen atom, R¹⁷ is independently H or —(CH₂)_(r)CH₃ respectively, R⁷, R¹⁰ and R¹³ are groups each independently selected from the group consisting of sulfur, oxygen, amide, imide, a divalent heterocycle consisting of 3 to 12 atoms and a cyclic hydrocarbon (Ar(R¹⁸ _(r)—R¹⁹)_(s)), R¹⁸ is —CR⁵R¹⁷, R¹⁹ is independently selected from the group consisting of H, —OH, —COOH, —C(NH₂)═NH, —CN, —SO₃H, —NH₂, —NHMe, —NMe₂ and a halogen atom; k, l, m, n, o, p and q are each independently 0 or an integer of 1 or more, r is 0 to 3, s is 0 to 12, and the sum total of k, l, m, n, o, p and q is 0 to 12); R² is a group selected from H, —OH, —NH₂, —NHMe, —NMe₂, —CN, and a lower alkyl group (which may be substituted with one or a plurality of polar groups); R⁴ is a group selected from H, —OH, —NH₂, —NHMe, —NMe₂, —CN, —SO₃H, a halogen and a lower alkyl group which may be optionally substituted with one or a plurality of polar groups; and j is 0 or 1 provided that, when j is 0, R¹ is not H and when j is 1, both of R¹ and R³ cannot be H.
 19. The compound having affinity with a calcified tissue according to claim 1, wherein the group AC having affinity with a calcified tissue comprises an organic phosphonic acid, and the organic phosphonic acid is an organic aminophosphonic acid derivative having an amine nitrogen atom to which a group represented by the formula II is bonded, or an ester or a salt thereof:

wherein t is an integer of 1 to 8; X and Y are each independently selected from hydrogen, a halogen group, a hydroxyl group, a carboxyl group, a carbonyl group, a phosphonic acid group, and a hydrocarbon group having 1 to 8 carbon atoms, and when t is larger than 1, each X and Y may be the same or different; R²⁰ is selected from hydrogen, a silyl group, an alkyl group, a benzyl group, sodium and potassium.
 20. The compound having affinity with a calcified tissue according to claim 1, wherein the group AC having affinity with a calcified tissue comprises an organic phosphonic acid, and the organic phosphonic acid is a phosphonic acid derivative represented by the formula III, an ester or a salt thereof.

wherein each u and u′ is independently an integer of 0 to 5, preferably 0, 1, or 2; R²¹, R²² and R²³ are each independently —(CH₂)_(v)— wherein v=1 to 5; A, B, C, D, E, and F are each independently selected from the group consisting of hydrogen, a methyl group, an ethyl group, an isopropyl group, a pivaloyl group, a benzyl group, an acetyl group, a trifluoroacetyl group, and groups of the following formulae IV-1 to 3, and one of A, B, C, D, E and F is the group of following formula IV-1.

wherein t, X and Y are the same as in the above-mentioned formula II; t′ is 2 or 3; X′ and Y′ are each independently selected from hydrogen, a methyl group and an ethyl group, and each X′ and Y′ may be the same or different.
 21. The compound having affinity with a calcified tissue according to claim 1, wherein the ligand (LI) for binding to a metal atom has a coordinating atom selected from oxygen, sulfur, phosphorus, nitrogen and carbon.
 22. The compound having affinity with a calcified tissue according to claim 1, wherein the ligand (LI) for binding to a metal atom is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), triethylenetetraaminehexaacetic acid (TTHA), cyclam, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N{1-2,3-dioleyloxy}propyl}-N,N,N-triethylammonium (DOTMA), mercaptoacetylglycylglycine (MAG3), ethylene cysteine dimer (ECD), hydrazinonicotinyl (HYNIC), lysine-tyrosine-cysteine (KYC), cysteine-glycine-cysteine (CYC), N,N′-bis(mercaptoacetamide)ethylenediamine (DADS), N,N′-bis(mercaptoacetamide)-2,3-diamine propanoic acid (CO2DADS), N,N′-bis(2-mercaptoethyl)ethylenediamine (BATs), thiosemicarbazone, propylene amineoxime (PnAO), and other amineoxime ligands and derivatives thereof.
 23. The compound having affinity with a calcified tissue according to claim 1, wherein the AC or LI has a linker L through which the AC or LI is coupled with the mother nucleus MC.
 24. The compound having affinity with a calcified tissue according to claim 22, wherein the linker L is selected from the group consisting of peptide, alkyl, and alkyl ether, alkylamide, alkylamine and alkylolefin represented by formula —(CH₂)_(w)—R²⁴—(CH₂)_(w)— (wherein w is each independently 0 to 5, and R²⁴ is O, S, NHCO, NH, or CH═CH).
 25. The compound having affinity with a calcified tissue according to claim 1, which is represented by the following formula V-I or V-2:

wherein R and R′ are each independently a group AC having affinity with a calcified tissue or a ligand LI for binding to a metal atom, and at least one of them is the group AC having affinity with a calcified tissue; x and y are each independently 0 to 19; and x+y is 1 to
 19. 26. The compound having affinity with a calcified tissue, represented by the following formula VI-1:


27. The compound having affinity with a calcified tissue, represented by the following formula VI-2:


28. The compound having affinity with a calcified tissue, represented by the following formula VII-1:


29. The compound having affinity with a calcified tissue, represented by the following formula VII-2:


30. The compound having affinity with a calcified tissue according to claim 25, which forms a complex with a metal atom.
 31. The compound having affinity with a calcified tissue according to claim 1, wherein the metal atom which forms a complex or a metal atom or isotope element contained in the mother nucleus MC, the group AC having affinity with a calcified tissue or the ligand LI is an element selected from the group consisting of elements of atomic number 6-9, 15-17, 21-29, 31, 35, 37-44, 49, 50, 53, 56-70, 72-75, 81, 83 and
 85. 32. The compound having affinity with a calcified tissue according to claim 31, wherein the metal atom is radioactive, paramagnetic or X-ray impermeable.
 33. The compound having affinity with a calcified tissue according to claim 1, wherein the metal atom or isotope element is a radioactive nuclide selected from the group consisting of 11-C, 15-0,18-F, 32-P, 59-Fe, 67-Cu, 67-Ga, 81-Rb, 89-Sr, 90-Y, 99m-Tc, 111-In, 123-I, 124-I, 125-I, 131-I, 117m-Sn, 153-Sm, 186-Re, 188-Re, 201-Ti, 211-At, 212-Bi and 213-Bi.
 34. The compound having affinity with a calcified tissue according to claim 1, wherein the metal atom or isotope element is an element selected from the group consisting of chromium (III), manganese (II), iron (II), iron (III), praseodymium (III), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), terbium(III), dysprosium (III), holmium (III), and erbium (III).
 35. The compound having affinity with a calcified tissue according to claim 1, wherein the metal atom or isotope element is an element selected from the group consisting of bismuth, tungsten, tantalum, hafnium, lanthanum, lanthanide, barium, molybdenum, niobium, zirconium and strontium.
 36. The compound having affinity with a calcified tissue according to claim 1, which is in a form of a salt, a hydrate, a solvate, an aggregate, an aqueous solution or a lyophilized product.
 37. The compound having affinity with a calcified tissue according to claim 1, wherein the particle size is 1 nm to 50 μm.
 38. A composition for producing a complex compound having affinity with a calcified tissue, which comprises a compound having affinity with a calcified tissue according to claim 1, a peroxide ion of a transition metal, and a reducing agent.
 39. A therapeutic agent which comprises a compound having affinity with a calcified tissue according to claim
 1. 40. A pharmaceutical composition which comprises a compound having affinity with a calcified tissue according to claim 1 or a salt thereof and at least one pharmacologically acceptable carrier.
 41. A kit for preparing a radioactive labeled compound, which comprises a compound having affinity with a calcified tissue according to claim
 1. 42. A diagnostic agent, imaging agent or therapeutic agent, which comprises a compound having affinity with a calcified tissue according to claim
 1. 43. A radioactive labeled compound diagnostic agent, imaging agent or therapeutic agent, which comprises a compound having affinity with a calcified tissue according to claim 33, a salt or an aggregate thereof.
 44. A nuclear magnetic resonance imaging agent which comprises a compound having affinity with a calcified tissue according to claim 34, a salt or an aggregate thereof.
 45. An X-ray imaging agent which comprises a compound having affinity with a calcified tissue according to claim 35, a salt or an aggregate thereof.
 46. A method of selectively modifying an amino group at a terminal end, which comprises providing an amino oligosaccharide having 2 to 50 saccharide units which consists of one or more monosaccharides selected from the group consisting of glucosamine, mannosamine and galactosamine and is reduced at a terminal end thereof, and subjecting the amino oligosaccharide to a reaction for generating a carbamate compound.
 47. A method of selectively modifying an amino group at a terminal end with a butoxycarbonyl (Boc) group, which comprises reacting, dibutyl dicarbonate, an aminosaccharide of 2 to 13 saccharide units which consists of one or more monosaccharides selected from the group consisting of glucosamine, mannosamine and galactosamine and reduced at a terminal end thereof. 